Novel immunoglobulin insertions, deletions, and substitutions

ABSTRACT

An Fc variant of a parent Fc polypeptide, wherein said Fc variant exhibits altered binding to one or more FcγRs, wherein said Fc variant comprises at least one amino acid insertion in the Fc region of said parent Fc polypeptide.

This application is a continuation of U.S. application Ser. No.12/020,443, filed Jan. 25, 2008, U.S. Ser. No. 12/020,443 claims benefitunder 35 U.S.C. §119(e) to USSNs 60/886,635, filed Jan. 25, 2007; and iscontination-in-part of U.S. Ser. No. 11/396,495, filed Mar. 31, 2006; acontinuation-in-part of U.S. Ser. No. 11/256,060, filed Oct. 21, 2005;U.S. Ser. No. 11/256,060 claims benefit under 35 U.SC. §119(e) to U.S.Ser. No. 60/659,004, filed Mar. 3, 2005; U.S. Ser. No. 60/652,968, filedFeb. 14, 2005; U.S. Ser. No. 60/629,068, filed Nov. 18, 2004; and U.S.Ser. No. 60/621,387, filed Oct. 21, 2004, all of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to novel immunoglobulin insertions,deletions, and substitutions that provide optimized effector functionproperties, engineering methods for their generation, and theirapplication, particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that bind a specific antigen.Generally, antibodies are specific for targets, have the ability tomediate immune effector mechanisms, and have a long half-life in serum.Such properties make antibodies powerful therapeutics. Monoclonalantibodies are used therapeutically for the treatment of a variety ofconditions including cancer, infectious disease, autoimmune disease, andinflammatory disorders. There are currently over ten antibody productson the market and hundreds in development.

Antibodies have found widespread application in oncology, particularlyfor targeting cellular antigens selectively expressed on tumor cellswith the goal of cell destruction. There are a number of mechanisms bywhich antibodies destroy tumor cells, including anti-proliferation viablockage of needed growth pathways, intracellular signaling leading toapoptosis, enhanced down regulation and/or turnover of receptors, CDC,ADCC, ADCP, and promotion of an adaptive immune response (Cragg et al.,1999, Curr Opin Immunol 11:541-547; Glennie et al., 2000, Immunol Today21:403-410, both hereby entirely incorporated by reference). Anti-tumorefficacy may be due to a combination of these mechanisms, and theirrelative importance in clinical therapy appears to be cancer dependent.

Despite this arsenal of anti-tumor weapons, the potency of antibodies asanti-cancer agents is unsatisfactory, particularly given their highcost. Patient tumor response data show that monoclonal antibodiesprovide only a small improvement in therapeutic success over normalsingle-agent cytotoxic chemotherapeutics. For example, just half of allrelapsed low-grade non-Hodgkin's lymphoma patients respond to theanti-CD20 antibody rituximab (McLaughlin et al., 1998, J Clin Oncol16:2825-2833, hereby entirely incorporated by reference). Of 166clinical patients, 6% showed a complete response and 42% showed apartial response, with median response duration of approximately 12months. Trastuzumab (Herceptin™, Genentech), an anti-HER2/neu antibodyfor treatment of metastatic breast cancer, has less efficacy. Theoverall response rate using trastuzumab for the 222 patients tested wasonly 15%, with 8 complete and 26 partial responses and a median responseduration and survival of 9 to 13 months (Cobleigh et al., 1999, J ClinOncol 17:2639-2648, hereby entirely incorporated by reference).Currently for anticancer therapy, any small improvement in mortalityrate defines success. Thus there is a significant need to enhance thecapacity of antibodies to destroy targeted cancer cells.

One potential way to improve the activity of anti-cancer therapeutics isto optimize their affinity and/or selectivity for Fc gamma receptors(FcγRs). Because all FcγRs interact with the same binding site on Fc,and because of the high homology among the FcγRs, obtaining variantsthat selectively increase or reduce FcγR affinity is a major challenge.Thus there is a need to make Fc variants that selectively increase orreduce FcγR affinity.

In contrast to antibody therapeutics and indications wherein effectorfunctions contribute to clinical efficacy, for some antibodies andclinical applications it may be favorable to reduce or eliminate bindingto one or more FcγRs, or reduce or eliminate one or more FcγR- orcomplement-mediated effector functions including but not limited toADCC, ADCP, and/or CDC. This is often the case for therapeuticantibodies whose mechanism of action involves blocking or antagonism butnot killing of the cells bearing target antigen. In these casesdepletion of target cells is undesirable and can be considered a sideeffect. For example, the ability of anti-CD4 antibodies to block CD4receptors on T cells makes them effective anti-inflammatories, yet theirability to recruit FcγR receptors also directs immune attack against thetarget cells, resulting in T cell depletion (Reddy et al., 2000, JImmunol 164:1925-1933, incorporated entirely by reference). Effectorfunction may also be a problem for radiolabeled antibodies, referred toas radioconjugates, and antibodies conjugated to toxins, referred to asimmunotoxins. These drugs can be used to destroy cancer cells, but therecruitment of immune cells via Fc interaction with FcγRs brings healthyimmune cells in proximity to the deadly payload (radiation or toxin),resulting in depletion of normal lymphoid tissue along with targetedcancer cells (Hutchins et al., 1995, Proc Natl Acad Sci USA92:11980-11984; White et al., 2001, Annu Rev Med 52:125-145, bothincorporated entirely by reference). What is needed is a general androbust means to completely ablate all FcγR binding and FcγR- andcomplement-mediated effector functions. These and other needs areaddressed by the present invention.

SUMMARY OF THE INVENTION

The present invention is directed to an Fc variant of a parent Fcpolypeptide, wherein said Fc variant comprises amino modifications,which can comprise independently or in combination amino acidinsertion(s), amino acid deletion(s) and/or amino acid substitutions,particularly in the Fc region of said parent Fc polypeptide.

In one aspect of the invention, the Fc variant of the inventioncomprises an amino acid insertion after a position selected from thegroup consisting of 233, 234, 235, 236, and 237, wherein numbering isaccording to the EU index. The Fc variant may additionally comprise anamino acid substitution in the Fc region. In one embodiment, saidsubstitution occurs at a position selected from the group consisting of235, 236, 237, 325, and 328, wherein numbering is according to the EUindex. In a preferred embodiment, said substitution is selected from thegroup consisting of 235G, 236R, 237K, 325L, 325A, and 328R. In anotherembodiment, said substitution occurs at a position selected from thegroup consisting of 234, 235, 236, 239, 243, 247, 255, 267, 268, 270,280, 292, 293, 295, 298, 300, 305, 324, 326, 327, 328, 330, 332, 333,334, 392, 396, and 421, wherein numbering is according to the EU index.In a preferred embodiment, said substitution is selected from the groupconsisting of 234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D,239E, 243L, 247L, 255L, 267D, 267E, 267Q, 268D, 268E, 270E, 280H, 280Q,280Y, 292P, 293R, 295E, 298A, 298T, 298N, 300L, 305I, 324G, 324I, 326A,326D, 326E, 326W, 326Y, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, A330V,332D, 332E, 333A, 333S, 334A, 334L, 392T, 396L, and 421K.

In another aspect of the invention, the Fc variant of the inventioncomprises an amino acid deletion at a position selected from the groupconsisting of 233, 234, 235, 236, and 237, wherein numbering isaccording to the EU index. The Fc variant may additionally comprise anamino acid substitution in the Fc region. In one embodiment, saidsubstitution occurs at a position selected from the group consisting of235, 236, 237, 325, and 328, wherein numbering is according to the EUindex. In a preferred embodiment, said substitution is selected from thegroup consisting of 235G, 236R, 237K, 325L, 325A, and 328R. In anotherembodiment, said substitution occurs at a position selected from thegroup consisting of 234, 235, 236, 239, 243, 247, 255, 267, 268, 270,280, 292, 293, 295, 298, 300, 305, 324, 326, 327, 328, 330, 332, 333,334, 392, 396, and 421, wherein numbering is according to the EU index.In a preferred embodiment, said substitution is selected from the groupconsisting of 234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D,239E, 243L, 247L, 255L, 267D, 267E, 267Q, 268D, 268E, 270E, 280H, 280Q,280Y, 292P, 293R, 295E, 298A, 298T, 298N, 300L, 305I, 324G, 324I, 326A,326D, 326E, 326W, 326Y, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, A330V,332D, 332E, 333A, 333S, 334A, 334L, 392T, 396L, and 421K.

In a preferred embodiment of the invention, the Fc variant altersbinding to one or more FcγRs. In one aspect, said Fc variant reducesaffinity to a human FcγR. In another aspect, said Fc variant improvesaffinity to a human FcγR.

The present invention provides novel Fc polypeptides, includingantibodies, Fc fusions, isolated Fc, and Fc fragments, that comprise theFc variants disclosed herein. The novel Fc polypeptides may find use ina therapeutic product. In certain embodiments, the Fc polypeptides ofthe invention are antibodies.

In one aspect of the invention, the Fc variant of the invention composesan antibody that is a human IgG1, IgG2, or IgG4 antibody.

The present invention provides isolated nucleic acids encoding the Fcvariants described herein. The present invention provides vectorscomprising the nucleic acids, optionally, operably linked to controlsequences. The present invention provides host cells containing thevectors, and methods for producing and optionally recovering the Fcvariants.

The present invention provides compositions comprising Fc polypeptidesthat comprise the Fc variants described herein, and a physiologically orpharmaceutically acceptable carrier or diluent.

The present invention contemplates therapeutic and diagnostic uses forFc polypeptides that comprise the Fc variants disclosed herein. The Fcpolypeptides described by the invention may be used to treat a varietyof indications, including but not limited to cancers, infectiousdiseases, autoimmune disorders, an infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 [SEQ ID NOS. 1-4]. Alignment of the amino acid sequences of thehuman IgG immunoglobulins IgG1, IgG2, IgG3, and IgG4. FIG. 1 a providesthe sequences of the CH1 (Cγ1) and hinge domains, and FIG. 1 b providesthe sequences of the CH2 (Cγ2) and CH3 (Cγ3) domains. Positions arenumbered according to the EU index of the IgG1 sequence, and differencesbetween IgG1 and the other immunoglobulins IgG2, IgG3, and IgG4 areshown in gray. Allotypic polymorphisms exist at a number of positions,and thus slight differences between the presented sequences andsequences in the prior art may exist. The possible beginnings of the Fcregion are labeled, defined herein as either EU position 226 or 230.

FIGS. 2 a and 2 b. Common haplotypes of the human gamma1 (FIG. 2 a) andgamma2 (FIG. 2 b) chains.

FIGS. 3 a and 3 b. FIG. 3 a provides an illustration of the hinge regionand sites of engineering. Gray indicates the C-terminus of the CH1domain (left) and N-terminus of the CH2 domain (right). Bold indicatesresidues in the hinge and N-terminal CH2 domain, 233-238, that interactwith FcγRs according to the structure of the human Fc/FcγRIIIb complex(pdb 1E4K, Sondermann et al., 2000, Nature 406:267-273). FIG. 3 b showsthe structured domains (CH2 and CH3) of the Fc region (pdb 1 DN2, DeLanoet al., 2000, Science 287:1279-1283). The first residues involved in thestructured CH2 region, G237 and P238, are shown as black sticks. Thecarbohydrate attached at N297 is shown as black lines.

FIG. 4. Library of antibody Fc variants screened for reduced FcγRaffinity and effector function. # indicates a deletion of the designatedresidue, and ̂ indicates an insertion of the designated amino acid afterthe designated position. A description of insertions and deletions isprovided for each variant, and the amino acid sequence from EU positions230-238 is provided.

FIGS. 5 a and 5 b. Surface Plasmon Resonance (SPR) (Biaore) sensorgramsfor binding of WT and Fc variant anti-Her2 antibodies to human Fcreceptors. FIG. 5 a shows the binding of anti-Her2 WT IgG1 antibody tohuman FcγRs FcγRI, H131 and R131 FcγRIIa, FcγRIIb, and V158 and F158FcγRIIIa. Binding was measured at 5 concentrations of receptor. FIG. 5 bshows the sensorgram for the highest receptor concentration for bindingof select antibodies and antibody variants to each FcγR.

FIG. 6. Table of affinities for binding of WT IgG and Fc variantantibodies to human FcγRs as determined by Biacore. The equilibriumdissociation constant (K_(D)) for binding of each variant to each FcγRis provided where tested. “NB”=no binding detected; “Weak”=bindingobserved but not fittable to an accurate K_(D). A blank cell indicatesthat the receptor was not tested for that particular variant.

FIG. 7. Affinities for binding of WT and Fc variant antibodies to humanFcγRs obtained from the data provided in FIG. 6. The graph is a plot ofthe log of the K_(A) (K_(A)=1/K_(D) as provided in FIG. 6) for bindingof each variant to each of the Fc receptors. I=FcγRI, H IIa=H131FcγRIIa, R IIa=R131 FcγRIIa, IIb=FcγRIIb, V IIIa=V158 FcγRIIIa, and FIIIa=F158 FcγRIIIa.

FIG. 8. SPR sensorgrams at the highest receptor concentration forbinding of WT and Fc variant antibodies to human FcγRI.

FIG. 9. SPR sensorgrams at the highest receptor concentration forbinding of WT and Fc variant antibodies to human FcγRI, H131 FcγRIIa,R131FcγRIIa, FcγRIIb, V158 FcγRIIIa, and F158 FcγRIIIa.

FIG. 10. ADCC assay comparing PBMC ADCC activity of anti-Her2 Fc variantantibodies with that of native IgG isotypes IgG1, IgG2, and IgG4.

FIG. 11. ADCP assay comparing macrophage phagocytosis of anti-CD19 Fcvariant antibody with that of native IgG1.

FIG. 12. CDC assay comparing complement activity of anti-CD20 Fc variantantibodies with that of native IgG isotypes IgG1, IgG2, and IgG4.

FIGS. 13 a and 13 b. Preferred modifications of the invention forreducing FcγR- and/or complement-mediated effector function. FIG. 13 ashows positions at which insertions and deletions may be constructed.FIG. 13 b shows positions and substitutions that may be combined withthe modifications provided in FIG. 13 a. However, as outlined herein,FIG. 13 is not meant to be limiting, and any amino acid modificationdescribed herein or in the applications incorporated by reference can becombined independently with any other(s).

FIG. 14. Library of antibody Fc variants screened for selective FcγRaffinity and optimized effector function. # indicates a deletion of thedesignated residue, and ̂ indicates an insertion of the designated aminoacid after the designated position. A description of insertions anddeletions is provided for each variant, and the amino acid sequence from230-238 is provided.

FIGS. 15 a and 15 b. FIG. 15 a provides the affinities for binding of WTIgG and Fc variant antibodies to human FcγRs as determined by Biacore.The equilibrium dissociation constant (K_(D)) for binding of eachvariant to each FcγR is provided where tested. “NB”=no binding detected.FIG. 15 b provides the activating:inhibitory ratios for two activatingreceptors, FcγRIIa (H131 and R131 isoforms) and FcγRIIIa (V1158 and F158isoforms) relative to the inhibitory receptor FcγRIIb. These ratios werecalculated by dividing the K_(D) for FcγRIIb by the K_(D) for theactivating receptor.

FIG. 16. Affinities for binding of WT and Fc variant antibodies to humanFcγRs obtained from SPR data provided in FIG. 15. The graph is a plot ofthe log of the K_(A) (K_(A)=1/K_(D) as provided in FIG. 15) for bindingof each variant to each of the Fc receptors. I=FcγRI, H IIa=H131FcγRIIa, R IIa=R131FcγRIIa, IIb=FcγRIIb, V IIIa=V158 FcγRIIIa, and FIIIa=F158 FcγRIIIa. ELLG=P233E/V234L/A235L/A235G.

FIG. 17. Affinity ratios of WT and Fc variant antibodies for the humanFcγRs. Data are provided in FIG. 15 b. ELLG=P233E/V234L/A235L/A235G.

FIGS. 18 a and 18 b. Preferred modifications of the invention forengineering selectively optimized FcγR affinity. FIG. 18 a showspositions at which insertions and deletions may be constructed. FIG. 18b shows positions and substitutions that may be combined with themodifications provided in FIG. 18 a.

FIGS. 19 a-19 f [SEQ ID NOS. 5-10]. Amino acid sequences of variablelight (VL) and heavy (VH) chains used in the present invention,including PRO70769 (FIGS. 19 a and 19 b), trastuzumab (FIGS. 19 c and 19d), and ipilimumab (FIGS. 19 e and 19 f).

FIGS. 20 a-20 e [SEQ ID NOS. 11-15. Amino acid sequences of humanconstant light kappa (FIG. 20 a) and heavy (FIGS. 20 b-20 e) chains usedin the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to proteins comprising altered Fcregions that exhibit altered functionality, including differentialbinding to one or more Fcγ receptors as compared to a non-altered Fcregion. In a particular embodiment, the variants reduce functionality,leading to desirable biological properties. The variants can include oneor more insertions of an amino acid, one or more deletions, and/or oneor more amino acid substitutions, as outlined herein.

Specifically, amino acid variations outlined in U.S. Ser. No.10/672,280, 10/822,231, 11/124,620, 11/174,287, 11/396,495; 11/538,406,11/538,411 and 60/886,635 include a variety of disclosures, all of whichare expressly incorporated by reference herein, and in particular forthe disclosure of positions, particular substitutions, data and thefigures.

In addition, each modification outlined herein can be done independentlyor in combination with any other modification(s), and can be done on theFc region of any or all of IgG1, IgG2, IgG3 and/or IgG4.

DEFINITIONS

In order that the invention may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “antibody” herein is meant a protein consisting of one or morepolypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (υ), delta (δ), gamma (γ), sigma (σ), and alpha(α) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE,and IgA (IgA1 and IgA2) isotypes respectively. Antibody herein is meantto include full length antibodies and antibody fragments, and may referto a natural antibody from any organism, an engineered antibody, or anantibody generated recombinantly for experimental, therapeutic, or otherpurposes. Additional description and definition of “antibody”, includingfor example “humanized”, “chimeric”, etc., is outlined below.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position.

By “CDC” or “complement dependent cytotoxicity” as used herein is meantthe reaction wherein one or more complement protein components recognizebound antibody on a target cell and subsequently cause lysis of thetarget cell.

By “constant region” of an antibody as defined herein is meant theregion of the antibody that is encoded by one of the light or heavychain immunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Cκ) or lambda (Cλ) light chains. Theconstant light chain typically comprises a single domain, and as definedherein refers to positions 108-214 of Cκ or Cλ, wherein numbering isaccording to the EU index. By “constant heavy chain” or “heavy chainconstant region” as used herein is meant the region of an antibodyencoded by the mu, delta, gamma, alpha, or epsilon genes to define theantibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For fulllength IgG antibodies, the constant heavy chain, as defined herein,refers to the N-terminus of the CH1 domain to the C-terminus of the CH3domain, thus comprising positions 118-447, wherein numbering isaccording to the EU index.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include FcγR-mediated effectorfunctions such as ADCC and ADCP, and complement-mediated effectorfunctions such as CDC.

By “effector cell” as used herein is meant a cell of the immune systemthat expresses one or more Fc receptors and mediates one or moreeffector functions. Effector cells include but are not limited tomonocytes, macrophages, neutrophils, dendritic cells, eosinophils, mastcells, platelets, B cells, large granular lymphocytes, Langerhans'cells, natural killer (NK) cells, and γδ T cells, and may be from anyorganism including but not limited to humans, mice, rats, rabbits, andmonkeys.

By “Fab” or “Fab region” as used herein is meant the polypeptides thatcomprise the V_(H), CH1, V_(H), and C_(L) immunoglobulin domains. Fabmay refer to this region in isolation, or this region in the context ofa full length antibody or antibody fragment.

By “Fc” or “Fc region” or “Fc domain”, as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain. Thus Fc refers to the lasttwo constant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. Fc may refer to this region inisolation, or this region in the context of an Fc polypeptide such as anantibody or immunoadhesin (e.g. an Fc fusion protein), as describedbelow. It should be noted that for the purposes described herein, “Fcregion” generally includes the hinge region, comprising residues230-238, unless noted otherwise. Thus, an “Fc variant” can includevariants of the hinge region, in the presence or absence of additionalamino acid modifications in the Cγ2 and Cγ3 domains.

By “Fc polypeptide” as used herein is meant a polypeptide that comprisesall or part of an Fc region. Fc polypeptides include antibodies, Fcfusions (sometimes referred to as “Fc fusion proteins” or“immunoadhesins”), isolated Fcs, and Fc fragments.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides is operably linked to Fc. Fc fusion is herein meant to besynonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”,and “receptor globulin” (sometimes with dashes) as used in the prior art(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immunol 9:195-200, both hereby entirely incorporated byreference). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general may be any protein, polypeptideor small molecule. The role of the non-Fc part of an Fc fusion, i.e.,the fusion partner, is to mediate target binding, and thus it isfunctionally analogous to the variable regions of an antibody. Virtuallyany protein or small molecule may be linked to Fc to generate an Fcfusion. Protein fusion partners may include, but are not limited to, thetarget-binding region of a receptor, an adhesion molecule, a ligand, anenzyme, a cytokine, a chemokine, or some other protein or proteindomain. Small molecule fusion partners may include any therapeutic agentthat directs the Fc fusion to a therapeutic target. Such targets may beany molecule, preferably an extracellular receptor that is implicated indisease.

By “Fc gamma receptor” or “FcγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,incorporated entirely by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32),FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs or FcγR isoforms or allotypes.

By “Fc ligand” or “Fc receptor” as used herein is meant a molecule,preferably a polypeptide, from any organism that binds to the Fc regionof an antibody to form an Fc-ligand complex. Fc ligands include but arenot limited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136). Fcligands may include undiscovered molecules that bind Fc.

By “full length antibody” as used herein is meant the structure thatconstitutes the natural biological form of an antibody, includingvariable and constant regions. For example, in most mammals, includinghumans and mice, the full length antibody of the IgG isotype is atetramer and consists of two identical pairs of two immunoglobulinchains, each pair having one light and one heavy chain, each light chaincomprising immunoglobulin domains VL and CL, and each heavy chaincomprising immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In somemammals, for example in camels and llamas, IgG antibodies may consist ofonly two heavy chains, each heavy chain comprising a variable domainattached to the Fc region.

By “IgG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this IgG comprises the subclasses or isotypesIgG1, IgG2, IgG3, and IgG4. In mice IgG comprises IgG1, IgG2a, IgG2b,IgG3.

By “immunoglobulin (Ig)” herein is meant a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes.Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains.

By “immunoglobulin (Ig) domain” as used herein is meant a region of animmunoglobulin that exists as a distinct structural entity asascertained by one skilled in the art of protein structure. Ig domainstypically have a characteristic β-sandwich folding topology. The knownIg domains in the IgG isotype of antibodies ar VH Cγ1, Cγ2, Cγ3, VL, andCL.

By “IgG” or “IgG immunoglobulin” as used herein is meant a polypeptidebelonging to the class of antibodies that are substantially encoded by arecognized immunoglobulin gamma gene. In humans this class comprises thesubclasses or isotypes IgG1, IgG2, IgG3, and IgG4. By “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Theknown human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1,IgA2, IgM, IgD, and IgE.

By “modification” herein is meant an alteration in the physical,chemical, or sequence properties of a protein, polypeptide, antibody, orimmunoglobulin. Preferred modifications of the invention are amino acidmodifications and glycoform modifications.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution 1332E refers to avariant polypeptide, in this case a constant heavy chain variant, inwhich the isoleucine at position 332 is replaced with glutamic acid. Itshould be noted that in some cases, the initial identification of thewild-type amino acid is not meant to be determinative; that is, “I332E”can also refer to a protein that contains an glutamic acid at position332, even if the wild-type amino acid in the particular parent proteinis not isoleucine. This can also be conveyed using “332E” language, forexample. In addition, as is generally noted herein, multiplemodifications, including multiple substitutions, can be made. In someembodiments, from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions are done, with any range within being contemplated, incombination with one or more insertion(s) and/or one or moredeletion(s).

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid at a particular position in a parentpolypeptide sequence. An insertion as described herein is designated bythe symbol “̂”, followed by the position, followed by the amino acid thatis inserted. For example, ̂236R designates an insertion of arginineafter postion 236; “̂236RR” depicts the insertion of two arginines afterposition 236, etc. For ease of reference, the original numbering afteran insertion is not changed; that is, in a molecule containing aninsertion, the amino acid normally found following the insertion site isstill numbered as if the insertion did not occur, unless notedotherwise.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid at a particular position in a parentpolypeptide sequence. A deletion as described herein is designated bythe symbol “#”, preceded by the amino acid and position that are to bedeleted. For example, G237# designates the deletion of glycine atposition 237. For ease of reference, the original numbering after adeletion is not changed; that is, in a molecule containing a deletion,the amino acid normally found following the deletion site is stillnumbered as if the deletion did not occur, unless noted otherwise.

As is noted herein, any amino acid modification outlined herein or inthe incorporated references can be combined with any other modification;the examples herein are not meant to be limiting. Thus, for example, itmay be desirable to combine one or more deletions with one or moreinsertions and one or more subsititutions; one or more deletions withone or more insertions; one or more deletions with one or moresubstitutions; one or more substitutions with one or more insertions,etc.

By “glycoform modification” or “modified glycoform” or “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to a protein, for example an antibody, wherein saidcarbohydrate composition differs chemically from that of a parentprotein. Modified glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus for example an Fc variant maycomprise a modified glycoform. Alternatively, modified glycoform mayrefer to the Fc variant that comprises the different carbohydrate oroligosaccharide. In particular, alterations in fucosylation areconsidered “engineered glycoforms”, as is more fully described below.

By “parent polypeptide”, “parent protein”, “precursor polypeptide”, or“precursor protein” as used herein is meant an unmodified polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent Fc polypeptide” as used herein is meant anFc polypeptide that is modified to generate a variant, and by “parentantibody” as used herein is meant an antibody that is modified togenerate a variant antibody. In some embodiments, the “parent” is awild-type protein.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. For example,position 297 is a position in the human antibody IgG1.

By “polypeptide” or “protein” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides and peptides.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant polypeptide”, “polypeptide variant”, or “variant” as usedherein is meant a polypeptide sequence that differs from that of aparent polypeptide sequence by virtue of at least one amino acidmodification. The parent polypeptide may be a naturally occurring orwild-type (WT) polypeptide, or may be a modified version of a WTpolypeptide. Variant polypeptide may refer to the polypeptide itself, acomposition comprising the polypeptide, or the amino sequence thatencodes it. Preferably, the variant polypeptide has at least one aminoacid modification compared to the parent polypeptide, e.g. from aboutone to about ten amino acid modifications, and preferably from about oneto about five amino acid modifications compared to the parent. Thevariant polypeptide sequence herein will preferably possess at leastabout 80% homology with a parent polypeptide sequence, and mostpreferably at least about 90% homology, more preferably at least about95% homology. Accordingly, by “Fc variant” or “variant Fc” as usedherein is meant an Fc sequence that differs from that of a parent Fcsequence by virtue of at least one amino acid modification. An Fcvariant may only encompass an Fc region, or may exist in the context ofan antibody, Fc fusion, isolated Fc, Fc fragment, or other polypeptidethat is substantially encoded by Fc. Fc variant may refer to the Fcpolypeptide itself, compositions comprising the Fc variant polypeptide,or the amino acid sequence that encodes it. By “Fc polypeptide variant”or “variant Fc polypeptide” as used herein is meant an Fc polypeptidethat differs from a parent Fc polypeptide by virtue of at least oneamino acid modification. By “protein variant” or “variant protein” asused herein is meant a protein that differs from a parent protein byvirtue of at least one amino acid modification. By “antibody variant” or“variant antibody” as used herein is meant an antibody that differs froma parent antibody by virtue of at least one amino acid modification. By“IgG variant” or “variant IgG” as used herein is meant an antibody thatdiffers from a parent IgG by virtue of at least one amino acidmodification. By “immunoglobulin variant” or “variant immunoglobulin” asused herein is meant an immunoglobulin sequence that differs from thatof a parent immunoglobulin sequence by virtue of at least one amino acidmodification.

By “wild type” or “WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein, polypeptide, antibody, immunoglobulin, IgG,etc. has an amino acid sequence or a nucleotide sequence that has notbeen intentionally modified.

Antibodies

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. The light and heavy chainvariable regions show significant sequence diversity between antibodies,and are responsible for binding the target antigen. Each chain is madeup of individual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. Heavy chains are classifiedas mu, delta, gamma, alpha, or epsilon, and define the antibody'sisotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has severalsubclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgAhas several subclasses, including but not limited to IgA1 and IgA2.Thus, “isotype” as used herein is meant any of the classes andsubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions. The known humanimmunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1,IgM2, IgD, and IgE.

Each of the light and heavy chains are made up of two distinct regions,referred to as the variable and constant regions. The IgG heavy chain iscomposed of four immunoglobulin domains linked from N- to C-terminus inthe order VH-CH1-CH2-CH3, referring to the heavy chain variable domain,heavy chain constant domain 1, heavy chain constant domain 2, and heavychain constant domain 3 respectively (also referred to asVH-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constantgamma 1 domain, constant gamma 2 domain, and constant gamma 3 domainrespectively). The IgG light chain is composed of two immunoglobulindomains linked from N- to C-terminus in the order VL-CL, referring tothe light chain variable domain and the light chain constant domainrespectively. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. The distinguishing features between these antibodyclasses are their constant regions, although subtler differences mayexist in the variable region.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.There are 6 CDRs total, three each per heavy and light chain, designatedVH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variableregion outside of the CDRs is referred to as the framework (FR) region.Although not as diverse as the CDRs, sequence variability does occur inthe FR region between different antibodies. Overall, this characteristicarchitecture of antibodies provides a stable scaffold (the FR region)upon which substantial antigen binding diversity (the CDRs) can beexplored by the immune system to obtain specificity for a broad array ofantigens. A number of high-resolution structures are available for avariety of variable region fragments from different organisms, someunbound and some in complex with antigen. Sequence and structuralfeatures of antibody variable regions are disclosed, for example, inMorea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods20:267-279, hereby entirely incorporated by reference, and the conservedfeatures of antibodies are disclosed, for example, in Maynard et al.,2000, Annu Rev Biomed Eng 2:339-376, hereby entirely incorporated byreference.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. In the IgG subclass ofimmunoglobulins, there are several immunoglobulin domains in the heavychain. By “immunoglobulin (Ig) domain” herein is meant a region of animmunoglobulin having a distinct tertiary structure. Of interest in thepresent invention are the heavy chain domains, including, the constantheavy (CH) domains and the hinge region. In the context of IgGantibodies, the IgG isotypes each have three CH regions. Accordingly,“CH” domains in the context of IgG are as follows: “CH1” refers topositions 118-220 according to the EU index as in Kabat. “CH2” refers topositions 237-340 according to the EU index as in Kabat, and “CH3”refers to positions 341-447 according to the EU index as in Kabat.

Another important region of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230 to 236.

Of particular interest in the present invention are the Fc regions. By“Fc” or “Fc region”, as used herein is meant the polypeptide comprisingthe constant region of an antibody excluding the first constant regionimmunoglobulin domain and in some cases, part of the hinge. Thus Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgG, and the last three constant region immunoglobulin domainsof IgE and IgM, and the flexible hinge N-terminal to these domains. ForIgA and IgM, Fc may include the J chain. For IgG, Fc comprisesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerhinge region between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. Fc may refer to this region in isolation, or this region inthe context of an Fc polypeptide, as described below. By “Fcpolypeptide” as used herein is meant a polypeptide that comprises all orpart of an Fc region. Fc polypeptides include antibodies, Fc fusions,isolated Fcs, and Fc fragments.

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG the Fc region, Fc comprisesIg domains Cγ2 and Cγ3 and the N-terminal hinge leading into Cγ2. Animportant family of Fc receptors for the IgG class are the Fc gammareceptors (FcγRs). These receptors mediate communication betweenantibodies and the cellular arm of the immune system (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu RevImmunol 19:275-290, both hereby entirely incorporated by reference). Inhumans this protein family includes FcγRI (CD64), including isoformsFcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa(including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 andFcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa(including allotypes V158 and F158) and FcγRIIIb (including allotypesFcγRIIIb-NA1 and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett82:57-65, hereby entirely incorporated by reference). These receptorstypically have an extracellular domain that mediates binding to Fc, amembrane spanning region, and an intracellular domain that may mediatesome signaling event within the cell. These receptors are expressed in avariety of immune cells including monocytes, macrophages, neutrophils,dendritic cells, eosinophils, mast cells, platelets, B cells, largegranular lymphocytes, Langerhans' cells, natural killer (NK) cells, andγγ T cells. Formation of the Fc/FcγR complex recruits these effectorcells to sites of bound antigen, typically resulting in signaling eventswithin the cells and important subsequent immune responses such asrelease of inflammation mediators, B cell activation, endocytosis,phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic andphagocytic effector functions is a potential mechanism by whichantibodies destroy targeted cells. The cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell isreferred to as antibody dependent cell-mediated cytotoxicity (ADCC)(Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie etal., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu RevImmunol 19:275-290, both hereby entirely incorporated by reference). Thecell-mediated reaction wherein nonspecific cytotoxic cells that expressFcγRs recognize bound antibody on a target cell and subsequently causephagocytosis of the target cell is referred to as antibody dependentcell-mediated phagocytosis (ADCP).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65, hereby entirely incorporated by reference). The FcγRs bind theIgG Fc region with different affinities: the high affinity binder FcγRIhas a Kd for IgG1 of 10.sup.−8 M.sup.−1, whereas the low affinityreceptors FcγRII and FcγRIII generally bind at 10.sup.−6 and 10.sup.−5respectively. The extracellular domains of FcγRIIIa and FcγRIIIb are 96%identical, however FcγRIIIb does not have a intracellular signalingdomain. Furthermore, whereas FcγRI, FcγRIIa/c, and FcγRIIIa are positiveregulators of immune complex-triggered activation, characterized byhaving an intracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM), FcγRIIb has an immunoreceptor tyrosine-basedinhibition motif (ITIM) and is therefore inhibitory. Thus the former arereferred to as activation receptors, and FcγRIIb is referred to as aninhibitory receptor. Despite these differences in affinities andactivities, all FcγRs bind the same region on Fc, at the N-terminal endof the Cγ2 domain and the preceding hinge. This interaction is wellcharacterized structurally (Sondermann et al., 2001, J Mol Biol309:737-749, hereby entirely incorporated by reference), and severalstructures of the human Fc bound to the extracellular domain of humanFcγRIIIb have been solved (pdb accession code 1E4K) (Sondermann et al.,2000, Nature 406:267-273, hereby entirely incorporated by reference)(pdb accession codes 1IIS and 1IIX) (Radaev et al., 2001, J Biol Chem276:16469-16477, hereby entirely incorporated by reference).

An overlapping but separate site on Fc serves as the interface for thecomplement protein C1q. In the same way that Fc/FcγR binding mediatesADCC, Fc/C1q binding mediates complement dependent cytotoxicity (CDC). Asite on Fc between the Cγ2 and Cγ3 domains mediates interaction with theneonatal receptor FcRn, the binding of which recycles endocytosedantibody from the endosome back to the bloodstream (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu RevImmunol 18:739-766, both hereby entirely incorporated by reference).This process, coupled with preclusion of kidney filtration due to thelarge size of the full length molecule, results in favorable antibodyserum half-lives ranging from one to three weeks. Binding of Fc to FcRnalso plays a key role in antibody transport. The binding site for FcRnon Fc is also the site at which the bacterial proteins A and G bind. Thetight binding by these proteins is typically exploited as a means topurify antibodies by employing protein A or protein G affinitychromatography during protein purification. The fidelity of theseregions, the complement and FcRn/protein A binding regions are importantfor both the clinical properties of antibodies and their development.

A key feature of the Fc region is the conserved N-linked glycosylationthat occurs at N297. This carbohydrate, or oligosaccharide as it issometimes referred, plays a critical structural and functional role forthe antibody, and is one of the principle reasons that antibodies mustbe produced using mammalian expression systems. Efficient Fc binding toFcγR and C1q requires this modification, and alterations in thecomposition of the N297 carbohydrate or its elimination affect bindingto these proteins (Umana et al., 1999, Nat Biotechnol 17:176-180; Davieset al., 2001, Biotechnol Bioeng 74:288-294; Mimura et al., 2001, J BiolChem 276:45539-45547; Radaev et al., 2001, J Biol Chem 276:16478-16483;Shields et al., 2001, J Biol Chem 276:6591-6604; Shields et al., 2002, JBiol Chem 277:26733-26740; Simmons et al., 2002, J Immunol Methods263:133-147, all hereby entirely incorporated by reference).

Variants of the Invention

In general, as outlined above and unless noted otherwise, Fc variantsinclude amino acid modifications in the hinge region and/or the Cγ2 andCγ3 regions.

An Fc variant comprises one or more amino acid modifications relative toa parent Fc polypeptide, wherein the amino acid modification(s)optionally provide one or more optimized properties, although in somecases, the variants exhibit substantially identical biologicalproperties. It should be recognized that “optimized” may includeincreases and/or decreases in biological activity. That is, as isoutlined herein, it may be desirable in some cases to substantiallyablate binding to one or more FcγRs, even an activating receptor such asFcγIIIa.

An Fc variant of the present invention differs in amino acid sequencefrom its parent IgG by virtue of at least one amino acid modification.Thus Fc variants of the present invention have at least one amino acidmodification compared to the parent. Alternatively, the Fc variants ofthe present invention may have more than one amino acid modification ascompared to the parent, for example from about one to fifty amino acidmodifications, preferably from about one to ten amino acidmodifications, and most preferably from about one to about five aminoacid modifications compared to the parent. Thus the sequences of the Fcvariants and those of the parent Fc polypeptide are substantiallyhomologous or identical. For example, the variant Fc variant sequencesherein will possess about 80% homology (including identity) with theparent Fc variant sequence, preferably at least about 90% homology, andmost preferably at least about 95, 96, 97, 98 and 99% identity.Modifications of the invention include amino acid modifications,including insertions, deletions, and substitutions. Modifications of theinvention also include glycoform modifications. Modifications may bemade genetically using molecular biology, or may be made enzymaticallyor chemically.

The Fc variants of the present invention are defined according to theamino acid modifications that compose them. Thus, for example, L328R isan Fc variant with the substitution L328R relative to the parent Fcpolypeptide. Likewise, ̂236R/L328R defines an Fc variant with theinsertion ̂236R and the substitution L328R relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as ̂236R/328R. Itis noted that the order in which modifications are provided isarbitrary, that is to say that, for example, ̂236R/L328R is the same Fcvariant as L328R/̂236R, and so on. For all positions discussed in thepresent invention, numbering is according to the EU index or EUnumbering scheme (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, hereby entirely incorporated byreference). The EU index or EU index as in Kabat or EU numbering schemerefers to the numbering of the EU antibody (Edelman et al., 1969, ProcNatl Acad Sci USA 63:78-85, hereby entirely incorporated by reference).

In one embodiment, one or more amino acid insertions are made. Aminoacid insertions can be made within the hinge region, including atpositions 233, 234, 235, 236 and 237. Exemplary insertions include, butare not limited to, ̂233L, ̂233EL, ̂234L, ̂235G, ̂235A, ̂235S, ̂235T,̂235N, ̂235D, ̂235V, ̂235L, ̂235R, ̂237R, ̂237RR, ̂297G, ̂297D, ̂297A,̂297S, ̂326G, ̂326T, ̂326D and ̂326E. Particular combinations ofinsertions and other modifications are also outlined in the figures. Allof these may be done in any IgG molecule, particularly in IgG1 and IgG2.In some embodiments, insertions of glycine after position 235 are notpreferred (̂235G), except in combinations with other amino acidmodifications.

In one embodiment, one or more amino acid deletions are made. Amino acidinsertions can be made within the hinge region, including at positions233, 234, 235, 236 and 237. Particular combinations of deletions andother modifications are also outlined in the figures. All of these maybe done in any IgG molecule, particularly in IgG1 and IgG2. In someembodiments, deletions at position 236 are not preferred (236#), exceptin combinations with other amino acid modifications.

In one embodiment, one or more amino acid substitutions are made. Aminoacid substitutions can be made at positions 221, 222, 224, 227, 228,230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244,245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288,290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318,320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,335 336 and 428, again, as any possible combination of substitution(s),insertion(s) and deletion(s). These amino acid substitutions include,but are not limited to, D221K, D221Y, K222E, K222Y, T223E, T223K, H224E,H224Y, T225E, T225K, T225W, P227E, P227G, P227K, P227Y, P228E, P228G,P228K, P228Y, P230A, P230E, P230G, P230Y, A231E, A231G, A231K, A231P,A231Y, P232E, P232G, P232K, P232Y, E233A, E233D, E233F, E233G, E233H,E233I, E233K, E233L, E233M, E233N, E233Q, E233R, E233S, E233T, E233V,E233W, E233Y, L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234K,L234M, L234N, L234P, L234Q, L234R, L234S, L234T, L234V, L234W, L234Y,L235A, L235D, L235E, L235F, L235G, L235H, L235I, L235K, L235M, L235N,L235P, L235Q, L235R, L235S, L235T, L235V, L235W, L235Y, G236A, G236D,G236E, G236F, G236H, G236I, G236K, G236L, G236M, G236N, G236P, G236Q,G236R, G236S, G236T, G236V, G236W, G236Y, G237D, G237E, G237F, G237H,G237I, G237K, G237L, G237M, G237N, G237P, G237Q, G237R, G237S, G237T,G237V, G237W, G237Y, P238D, P238E, P238F, P238G, P238H, P238I, P238K,P238L, P238M, P238N, P238Q, P238R, P238S, P238T, P238V, P238W, P238Y,S239D, S239E, S239F, S239G, S239H, S239I, S239K, S239L, S239M, S239N,S239P, S239Q, S239R, S239T, S239V, S239W, S239Y, V240A, V240I, V240M,V240T, F241D, F241E, F241L, F241R, F241S, F241W, F241Y, F243E, F243H,F243L, F243Q, F243R, F243W, F243Y, P244H, P245A, K246D, K246E, K246H,K246Y, P247G, P247V, D249H, D249Q, D249Y, R255E, R255Y, E258H, E258S,E258Y, T260D, T260E, T260H, T260Y, V262A, V262E, V262F, V262I, V262T,V263A, V263I, V263M, V263T, V264A, V264D, V264E, V264F, V264G, V264H,V264I, V264K, V264L, V264M, V264N, V264P, V264Q, V264R, V264S, V264T,V264W, V264Y, D265F, D265G, D265H, D265I, D265K, D265L, D265M, D265N,D265P, D265Q, D265R, D265S, D265T, D265V, D265W, D265Y, V266A, V266I,V266M, V266T, S267D, S267E, S267F, S267H, S267I, S267K, S267L, S267M,S267N, S267P, S267Q, S267R, S267T, S267V, S267W, S267Y, H268D, H268E,H268F, H268G, H268I, H268K, H268L, H268M, H268P, H268Q, H268R, H268T,H268V, H268W, E269F, E269G, E269H, E269I, E269K, E269L, E269M, E269N,E269P, E269R, E269S, E269T, E269V, E269W, E269Y, D270F, D270G, D270H,D270I, D270L, D270M, D270P, D270Q, D270R, D270S, D270T, D270W, D270Y,P271A, P271D, P271E, P271F, P271G, P271H, P271I, P271K, P271L, P271M,P271N, P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, E272D, E272F,E272G, E272H, E272I, E272K, E272L, E272M, E272P, E272R, E272S, E272T,E272V, E272W, E272Y, V273I, K274D, K274E, K274F, K274G, K274H, K274I,K274L, K274M, K274N, K274P, K274R, K274T, K274V, K274W, K274Y, F275L,F275W, N276D, N276E, N276F, N276G, N276H, N276I, N276L, N276M, N276P,N276R, N276S, N276T, N276V, N276W, N276Y, Y278D, Y278E, Y278G, Y278H,Y278I, Y278K, Y278L, Y278M, Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T,Y278V, Y278W, D280G, D280K, D280L, D280P, D280W, G281D, G281E, G281K,G281N, G281P, G281Q, G281Y, V282E, V282G, V282K, V282P, V282Y, E283G,E283H, E283K, E283L, E283P, E283R, E283Y, V284D, V284E, V284L, V284N,V284Q, V284T, V284Y, H285D, H285E, H285K, H285Q, H285W, H285Y, N286E,N286G, N286P, N286Y, K288D, K288E, K288Y, K290D, K290H, K290L, K290N,K290W, P291D, P291E, P291G, P291H, P291I, P291Q, P291T, R292D, R292E,R292T, R292Y, E293F, E293G, E293H, E293I, E293L, E293M, E293N, E293P,E293R, E293S, E293T, E293V, E293W, E293Y, E294F, E294G, E294H, E294I,E294K, E294L, E294M, E294P, E294R, E294S, E294T, E294V, E294W, E294Y,Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295M, Q295N, Q295P, Q295R,Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A, Y296D, Y296E, Y296G, Y296H,Y296I, Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S, Y296T, Y296V,N297D, N297E, N297F, N297G, N297H, N297I, N297K, N297L, N297M, N297P,N297Q, N297R, N297S, N297T, N297V, N297W, N297Y, S298D, S298E, S298F,S298H, S298I, S298K, S298M, S298N, S298Q, S298R, S298T, S298W, S298Y,T299A, T299D, T299E, T299F, T299G, T299H, T299I, T299K, T299L, T299M,T299N, T299P, T299Q, T299R, T299S, T299V, T299W, T299Y, Y300A, Y300D,Y300E, Y300G, Y300H, Y300K, Y300M, Y300N, Y300P, Y300Q, Y300R, Y300S,Y300T, Y300V, Y300W, R301D, R301E, R301H, R301Y, V302I, V303D, V303E,V303Y, S304D, S304H, S304L, S304N, S304T, V305E, V305T, V305Y, W313F,K317E, K317Q, E318H, E318L, E318Q, E318R, E318Y, K320D, K320F, K320G,K320H, K320I, K320L, K320N, K320P, K320S, K320T, K320V, K320W, K320Y,K322D, K322F, K322G, K322H, K322I, K322P, K322S, K322T, K322V, K322W,K322Y, V323I, S324D, S324F, S324G, S324H, S324I, S324L, S324M, S324P,S324R, S324T, S324V, S324W, S324Y, N325A, N325D, N325E, N325F, N325G,N325H, N325I, N325K, N325L, N325M, N325P, N325Q, N325R, N325S, N325T,N325V, N325W, N325Y, K326I, K326L, K326P, K326T, A327D, A327E, A327F,A327H, A327I, A327K, A327L, A327M, A327N, A327P, A327R, A327S, A327T,A327V, A327W, A327Y, L328A, L328D, L328E, L328F, L328G, L328H, L328I,L328K, L328M, L328N, L328P, L328Q, L328R, L328S, L328T, L328V, L328W,L328Y, P329D, P329E, P329F, P329G, P329H, P329I, P329K, P329L, P329M,P329N, P329Q, P329R, P329S, P329T, P329V, P329W, P329Y, A330E, A330F,A330G, A330H, A330I, A330L, A330M, A330N, A330P, A330R, A330S, A330T,A330V, A330W, A330Y, P331D, P331F, P331H, P331I, P331L, P331M, P331Q,P331R, P331T, P331V, P331W, P331Y, I332A, I332D, I332E, I332F, I332H,I332K, I332L, I332M, I332N, I332P, I332Q, I332R, I332S, I332T, I332V,I332W, I332Y, E333F, E333H, E333I, E333L, E333M, E333P, E333T, E333Y,K334F, K334I, K334L, K334P, K334T, T335D, T335F, T335G, T335H, T335I,T335L, T335M, T335N, T335P, T335R, T335S, T335V, T335W, T335Y, I336E,1336K, I336Y, S337E, S337H, and S337N, D221K, D221Y, K222E, K222Y,T223E, T223K, H224E, H224Y, T225E, T225K, T225W, P227E, P227G, P227K,P227Y, P228E, P228G, P228K, P228Y, P230A, P230A/E233D,P230A/E233D/I332E, P230E, P230G, P230Y, A231E, A231G, A231K, A231P,A231Y, P232E, P232G, P232K, P232Y, E233A, E233D, E233F, E233G, E233H,E233I, E233K, E233L, E233M, E233N, E233Q, E233R, E233S, E233T, E233V,E233W, E233Y, L234A, L234D, L234E, L234F, L234G, L234H, L234I,L234I/L235D, L234K, L234M, L234N, L234P, L234Q, L234R, L234S, L234T,L234V, L234W, L234Y, L235A, L235D, L235D/S239D/A330Y/I332E,L235D/S239D/N297D/I332E, L235E, L235F, L235G, L235H, L235I, L235K,L235M, L235N, L235P, L235Q, L235R, L235S, L235T, L235V, L235W, L235Y,G236A, G236D, G236E, G236F, G236H, G236I, G236K, G236L, G236M, G236N,G236P, G236Q, G236R, G236S, G236T, G236V, G236W, G236Y, G237D, G237E,G237F, G237H, G237I, G237K, G237L, G237M, G237N, G237P, G237Q, G237R,G237S, G237T, G237V, G237W, G237Y, P238D, P238E, P238F, P238G, P238H,P238I, P238K, P238L, P238M, P238N, P238Q, P238R, P238S, P238T, P238V,P238W, P238Y, S239D, S239D/A330L/I332E, S239D/A330Y/I332E/L234I,S239D/A330Y/I332E/V266I, S239D/D265F/N297D/I332E,S239D/D265H/N297D/I332E, S239D/D265I/N297D/I332E,S239D/D265L/N297D/I332E, S239D/D265T/N297D/I332E,S239D/D265Y/N297D/I332E, S239D/E272I/A330L/I332E, S239D/E272I/I332E,S239D/E272K/A330L/I332E, S239D/E272K/I332E, S239D/E272S/A330L/I332E,S239D/E272S/I332E, S239D/E272Y/A330L/I332E, S239D/E272Y/I332E,S239D/F241S/F243H/V262T/V264T/N297D/A330Y/I332E, S239D/H268D,S239D/H268E, S239D/I332D, S239D/I332E, S239D/I332E/A327D,S239D/I332E/A330I, S239D/I332E/A330Y, S239D/I332E/E272H,S239D/I332E/E272R, S239D/I332E/E283H, S239D/I332E/E283L,S239D/I332E/G236A, S239D/I332E/G236S, S239D/I332E/H268D,S239D/I332E/H268E, S239D/I332E/K246H, S239D/I332E/R255Y,S239D/I332E/S267E, S239D/I332E/V264I, S239D/I332E/V264I/A330L,S239D/I332E/V264I/S298A, S239D/I332E/V284D, S239D/I332E/V284E,S239D/I332E/V284E, S239D/I332N, S239D/I3320, S239D/K274E/A330L/I332E,S239D/K274E/I332E, S239D/K326E/A330L/I332E, S239D/K326E/A330Y/I332E,S239D/K326E/I332E, S239D/K326T/A330Y/I332E, S239D/K326T/I332E,S239D/N297D/A330Y/I332E, S239D/N297D/I332E, S239D/N297D/K326E/I332E,S239D/S267E/A330L/I332E, S239D/S267E/I332E, S239D/S298A/K326E/I332E,S239D/S298A/K326T/I332E, S239D/V240I/A330Y/I332E,S239D/V264T/A330Y/I332E, S239D/Y278T/A330L/I332E, S239D/Y278T/I332E,S239E, S239E/D265G, S239E/D265N, S239E/D265Q, S239E/I332D, S239E/I332E,S239E/I332N, S239E/I332Q, S239E/N297D/I332E, S239E/V264I/A330Y/I332E,S239E/V264I/I332E, S239E/V264I/S298A/A330Y/I332E, S239F, S239G, S239H,S239I, S239K, S239L, S239M, S239N, S239N/I332D, S239N/I332E,S239N/I332E/A330L, S239N/I332E/A330Y, S239N/I332N, S239N/I332Q, S239P,S239Q, S239Q/I332D, S239Q/I332E, S239Q/I332N, S239Q/I332Q,S239Q/V264I/I332E, S239R, S239T, S239V, S239W, S239Y, V240A, V240I,V240I/V266I, V240M, V240T, F241D, F241E, F241E/F243Q/V262T/V264E/I332E,F241E/F243Q/V262T/V264E, F241E/F243R/V262E/V264R/I332E,F241E/F243R/V262E/V264R, F241E/F243Y/V262T/V264R/I332E,F241E/F243Y/C262T/V264R, F241L, F241L/F243L/V262I/V264I, F241L/V262I,F241R/F243Q/V262T/V264R/I332E, F241R/F243Q/V262T/V264R, F241W,F241W/F243W, F241W/F243W/V262A/V264A, F241 Y,F241Y/F243Y/V262T/V264T/N297D/I332E, F241Y/F243Y/V262T/V264T, F243E,F243L, F243L/V262I/N264W, F243L/V264I, F243W, P244H, P244H/P245A/P247V,P245A, K246D, K246E, K246H, K246Y, P247G, P247V, D249H, D249Q, D249Y,R255E, R255Y, E258H, E258S, E258Y, T260D, T260E, T260H, T260Y, V262E,V262F, V263A, V263I, V263M, V263T, V264A, V264D, V264E,V264E/N297D/I332E, V264F, V264G, V264H, V264I, V264I/A330L/I332E,V264I/A330Y/I332E, V264I/I332E, V264K, V264L, V264M, V264N, V264P,V264Q, V264R, V264S, V264T, V264W, V264Y, D265F, D265F/N297E/I332E,D265G, D265H, D265I, D265K, D265L, D265M, D265N, D265P, D265Q, D265R,D265S, D265T, D265V, D265W, D265Y, D265Y/N297D/I332E,D265Y/N297D/T299L/I332E, V266A, V266I, V266M, V266T, S267D, S267E,S267E, S267E/A327D, S267E/P331D, S267E/S324I, S267E/V282G, S267F, S267H,S267I, S267K, S267L, S267L/A327S, S267M, S267N, S267P, S267Q,S267Q/A327S, S267R, S267T, S267V, S267W, S267Y, H268D, H268E, H268F,H268G, H268I, H268K, H268L, H268M, H268P, H268Q, H268R, H268T, H268V,H268W, E269F, E269G, E269H, E269I, E269K, E269L, E269M, E269N, E269P,E269R, E269S, E269T, E269V, E269W, E269Y, D270F, D270G, D270H, D270I,D270L, D270M, D270P, D270Q, D270R, D270S, D270T, D270W, D270Y, P271A,P271D, P271E, P271F, P271G, P271H, P271I, P271K, P271L, P271M, P271N,P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, E272D, E272F, E272G,E272H, E272I, E272K, E272L, E272M, E272P, E272R, E272S, E272T, E272V,E272W, E272Y, V273I, K274D, K274E, K274F, K274G, K274H, K274I, K274L,K274M, K274N, K274P, K274R, K274T, K274V, K274W, K274Y, F275L, F275W,N276D, N276E, N276F, N276G, N276H, N276I, N276L, N276M, N276P, N276R,N276S, N276T, N276V, N276W, N276Y, Y278D, Y278E, Y278G, Y278H, Y278I,Y278K, Y278L, Y278M, Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T, Y278V,Y278W, Y278W, Y278W/E283R/V302I, Y278W/V302I, D280G, D280K, D280L,D280P, D280W, G281D, G281D/V282G, G281E, G281K, G281N, G281P, G281Q,G281Y, V282E, V282G, V282G/P331D, V282K, V282P, V282Y, E283G, E283H,E283K, E283L, E283P, E283R, E283R/V302I/Y278W/E283R, E283Y, V284D,V284E, V284L, V284N, V284Q, V284T, V284Y, H285D, H285E, H285K, H285Q,H285W, H285Y, N286E, N286G, N286P, N286Y, K288D, K288E, K288Y, K290D,K290H, K290L, K290N, K290W, P291D, P291E, P291G, P291H, P291I, P291Q,P291T, R292D, R292E, R292T, R292Y, E293F, E293G, E293H, E293I, E293L,E293M, E293N, E293P, E293R, E293S, E293T, E293V, E293W, E293Y, E294F,E294G, E294H, E294I, E294K, E294L, E294M, E294P, E294R, E294S, E294T,E294V, E294W, E294Y, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295M,Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A, Y296D,Y296E, Y296G, Y296I, Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S,Y296T, Y296V, N297D, N297D/I332E, N297D/I332E/A330Y,N297D/I332E/S239D/A330L, N297D/I332E/S239D/D265V,N297D/I332E/S298A/A330Y, N297D/I332E/T299E, N297D/I332E/T299F,N297D/I332E/T299H, N297D/I332E/T299I, N297D/I332E/T299L,N297D/I332E/T299V, N297D/I332E/V296D, N297D/I332E/Y296E,N297D/I332E/Y296H, N297D/I332E/Y296N, N297D/I332E/Y296Q,N297D/I332E/Y296T, N297E/I332E, N297F, N297G, N297H, N297I, N297K,N297L, N297M, N297P, N297Q, N297R, N297S, N297S/I332E, N297T, N297V,N297W, N297Y, S298A/I332E, S298A/K326E, S298A/K326E/K334L, S298A/K334L,S298D, S298E, S298F, S298H, S298I, S298K, S298M, S298N, S298Q, S298R,S298T, S298W, S298Y, T299A, T299D, T299E, T299F, T299G, T299H, T299I,T299K, T299L, T299M, T299N, T299P, T299Q, T299R, T299S, T299V, T299W,T299Y, Y300A, Y300D, Y300E, Y300G, Y300H, Y300K, Y300M, Y300N, Y300P,Y300Q, Y300R, Y300S, Y300T, Y300V, Y300W, R301D, R301E, R301H, R301Y,V302I, V303D, V303E, V303Y, S304D, S304H, S304L, S304N, S304T, V305E,V305T, V305Y, W313F, K317E, K317Q, E318H, E318L, E318Q, E318R, E318Y,K320D, K320F, K320G, K320H, K320I, K320L, K320N, K320P, K320S, K320T,K320V, K320W, K320Y, K322D, K322F, K322G, K322H, K322I, K322P, K322S,K322T, K322V, K322W, K322Y, V323I, S324D, S324F, S324G, S324H, S324I,S324I/A327D, S324L, S324M, S324P, S324R, S324T, S324V, S324W, S324Y,N325A, N325D, N325E, N325F, N325G, N325H, N325I, N325K, N325L, N325M,N325P, N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326I, K326L,K326P, K326T, A327D, A327E, A327F, A327H, A327I, A327K, A327L, A327M,A327N, A327P, A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328D,L328D/I332E, L328E, L328E/I332E, L328F, L328G, L328H, L328H/I332E,L328I, L328I/I332E, L328I/I332E, L328K, L328M, L328M/I332E, L328N,L328N/I332E, L328P, L328Q, L328Q/I332E, L328Q/I332E, L328R, L328S,L328T, L328T/I332E, L328V, L328V/I332E, L328W, L328Y, P329D, P329E,P329F, P329G, P329H, P329I, P329K, P329L, P329M, P329N, P329Q, P329R,P329S, P329T, P329V, P329W, P329Y, A330E, A330F, A330G, A330H, A330I,A330L, A330L/I332E, A330M, A330N, A330P, A330R, A330S, A330T, A330V,A330W, A330Y, A330Y/I332E, P331D, P331F, P331H, P331I, P331L, P331M,P331Q, P331R, P331T, P331V, P331W, P331Y, I332A, I332D, I332E,I332E/G281D, I332E/H268D, I332E/H268E, I332E/S239D/S298A,I332E/S239N/S298A, I332E/V264I/S298A, I332E/V284E, I332F, I332H, I332K,I332L, I332M, I332N, I332P, I332Q, I332R, I332S, I332T, I332V, I332W,I332Y, E333F, E333H, E333I, E333L, E333M, E333P, E333T, E333Y, K334F,K334I, K334P, K334T, T335D, T335F, T335G, T335H, T335I, T335L, T335M,T335N, T335P, T335R, T335S, T335V, T335W, T335Y, I336E, I336K, I336Y,S337E, S337H, and S337N, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat, as is true throughout.Particular combinations of insertion(s), deletion(s) and othermodifications are also outlined in the figures. All of these may be donein any IgG molecule, particularly in IgG1 and IgG2.

In some embodiments, combinations of modifications that find use in thepresent invention are found in FIGS. 4 and 6-17, and additionallyinclude ̂236R/L328R (particularly in IgG1) and ̂236A/I332E (particularlyin IgG2). Similarly, amino modifications at 332 and/or 239 can becoupled with insertion(s) and/or deletion(s).

Functionally, variants that result in increased binding to activatingFcγRs as compared to the change in binding affinity to inhibitory FcγRsfind particular use in some embodiments.

The Fc variants of the present invention may be substantially encoded byimmunoglobulin genes belonging to any of the antibody classes. Incertain embodiments, the Fc variants of the present invention find usein antibodies or Fc fusions that comprise sequences belonging to the IgGclass of antibodies, including IgG1, IgG2, IgG3, or IgG4. FIG. 1provides an alignment of these human IgG sequences. In an alternateembodiment the Fc variants of the present invention find use inantibodies or Fc fusions that comprise sequences belonging to the IgA(including subclasses IgA1 and IgA2), IgD, IgE, IgG, or IgM classes ofantibodies. The Fc variants of the present invention may comprise morethan one protein chain. That is, the present invention may find use inan antibody or Fc fusion that is a monomer or an oligomer, including ahomo- or hetero-oligomer.

In certain embodiments, the Fc variants of the invention are based onhuman IgG sequences, and thus human IgG sequences are used as the “base”sequences against which other sequences are compared, including but notlimited to sequences from other organisms, for example rodent andprimate sequences. Fc variants may also comprise sequences from otherimmunoglobulin classes such as IgA, IgE, IgGD, IgGM, and the like. It iscontemplated that, although the Fc variants of the present invention areengineered in the context of one parent IgG, the variants may beengineered in or “transferred” to the context of another, second parentIgG. This is done by determining the “equivalent” or “corresponding”residues and substitutions between the first and second IgG, typicallybased on sequence or structural homology between the sequences of thefirst and second IgGs. In order to establish homology, the amino acidsequence of a first IgG outlined herein is directly compared to thesequence of a second IgG. After aligning the sequences, using one ormore of the homology alignment programs known in the art (for exampleusing conserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first Fc variant are defined. Alignment ofconserved residues preferably should conserve 100% of such residues.However, alignment of greater than 75% or as little as 50% of conservedresidues is also adequate to define equivalent residues. Equivalentresidues may also be defined by determining structural homology betweena first and second IgG that is at the level of tertiary structure forIgGs whose structures have been determined. In this case, equivalentresidues are defined as those for which the atomic coordinates of two ormore of the main chain atoms of a particular amino acid residue of theparent or precursor (N on N, CA on CA, C on C and O on O) are withinabout 0.13 nm and preferably about 0.1 nm after alignment. Alignment isachieved after the best model has been oriented and positioned to givethe maximum overlap of atomic coordinates of non-hydrogen protein atomsof the proteins. Regardless of how equivalent or corresponding residuesare determined, and regardless of the identity of the parent IgG inwhich the IgGs are made, what is meant to be conveyed is that the Fcvariants discovered by the present invention may be engineered into anysecond parent IgG that has significant sequence or structural homologywith the Fc variant. Thus for example, if a variant antibody isgenerated wherein the parent antibody is human IgG1, by using themethods described above or other methods for determining equivalentresidues, the variant antibody may be engineered in another IgG1 parentantibody that binds a different antigen, a human IgG2 parent antibody, ahuman IgA parent antibody, a mouse IgG2a or IgG2b parent antibody, andthe like. Again, as described above, the context of the parent Fcvariant does not affect the ability to transfer the Fc variants of thepresent invention to other parent IgGs.

Fc variants of the present invention may be substantially encoded bygenes from any organism, preferably mammals, including but not limitedto humans, rodents including but not limited to mice and rats,lagomorpha including but not limited to rabbits and hares, camelidaeincluding but not limited to camels, llamas, and dromedaries, andnon-human primates, including but not limited to Prosimians, Platyrrhini(New World monkeys), Cercopithecoidea (Old World monkeys), andHominoidea including the Gibbons and Lesser and Great Apes. In a certainembodiments, the Fc variants of the present invention are substantiallyhuman.

As is well known in the art, immunoglobulin polymorphisms exist in thehuman population. Gm polymorphism is determined by the IGHG1, IGHG2 andIGHG3 genes which have alleles encoding allotypic antigenic determinantsreferred to as G1 m, G2m, and G3m allotypes for markers of the humanIgG1, IgG2 and IgG3 molecules (no Gm allotypes have been found on thegamma 4 chain). Markers may be classified into ‘allotypes’ and‘isoallotypes’. These are distinguished on different serological basesdependent upon the strong sequence homologies between isotypes.Allotypes are antigenic determinants specified by allelic forms of theIg genes. Allotypes represent slight differences in the amino acidsequences of heavy or light chains of different individuals. Even asingle amino acid difference can give rise to an allotypic determinant,although in many cases there are several amino acid substitutions thathave occurred. Allotypes are sequence differences between alleles of asubclass whereby the antisera recognize only the allelic differences. Anisoallotype is an allele in one isotype which produces an epitope whichis shared with a non-polymorphic homologous region of one or more otherisotypes and because of this the antisera will react with both therelevant allotypes and the relevant homologous isotypes (Clark, 1997,IgG effector mechanisms, Chem Immunol. 65:88-110; Gorman & Clark, 1990,Semin Immunol 2(6):457-66, both hereby entirely incorporated byreference).

Allelic forms of human immunoglobulins have been well-characterized (WHOReview of the notation for the allotypic and related markers of humanimmunoglobulins. J Immunogen 1976, 3: 357-362; WHO Review of thenotation for the allotypic and related markers of human immunoglobulins.1976, Eur. J. Immunol. 6, 599-601; Loghem E van, 1986, Allotypicmarkers, Monogr Allergy 19: 40-51, all hereby entirely incorporated byreference). Additionally, other polymorphisms have been characterized(Kim et al., 2001, J. Mol. Evol. 54:1-9, hereby entirely incorporated byreference). At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) orG1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15,16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1, c5, u,v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis ofstructure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990);Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211, both hereby entirelyincorporated by reference). Allotypes that are inherited in fixedcombinations are called Gm haplotypes. FIG. 2 shows common haplotypes ofthe gamma chain of human IgG1 (FIG. 2 a) and IgG2 (FIG. 2 b) showing thepositions and the relevant amino acid substitutions. The Fc variants ofthe present invention may be substantially encoded by any allotype,isoallotype, or haplotype of any immunoglobulin gene.

The Fc variants of the invention can compose any Fc polypeptide,including but not limited to antibodies, isolated Fcs, Fc fragments, andFc fusions. In one embodiment, the Fc polypeptide of the invention is afull length antibody, constituting the natural biological form of anantibody, including variable and constant regions. For the IgG isotypefull length antibody is a tetramer and consists of two identical pairsof two immunoglobulin chains, each pair having one light and one heavychain, each light chain comprising immunoglobulin domains VL and CL, andeach heavy chain comprising immunoglobulin domains VH, Cyt, Cγ2, andCγ3.

Fc polypeptides and antibodies of the invention can be a variety ofstructures, including, but not limited antibody fragments, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, antibody fusions (sometimes referredto as “antibody conjugates”), and fragments of each, respectively.

In one embodiment, the antibody is an antibody fragment. Of particularinterest are antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment,which consists of a single variable, (v) isolated CDR regions, (vi)F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site, (viii) bispecificsingle chain Fv dimers, and (ix) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion. Theantibody fragments may be modified. For example, the molecules may bestabilized by the incorporation of disulphide bridges linking the VH andVL domains. Examples of antibody formats and architectures are describedin Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136, andCarter 2006, Nature Reviews Immunology 6:343-357 and references citedtherein, all expressly incorporated by reference.

In one embodiment, the antibody of the invention is a multispecificantibody, and notably a bispecific antibody, also sometimes referred toas “diabodies”. These are antibodies that bind to two (or more)different antigens. Diabodies can be manufactured in a variety of waysknown in the art, e.g., prepared chemically or from hybrid hybridomas.In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. In somecases, the scFv can be joined to the Fc region, and may include some orall of the hinge region. For a description of multispecific antibodiessee Holliger & Hudson, 2006, Nature Biotechnology 23(9):1126-1136 andreferences cited therein, all expressly incorporated by reference.

Fc Fusions, Antibody Fusions, and Antibody Conjugates

In addition to antibodies, an antibody-like protein that is finding anexpanding role in research and therapy is the Fc fusion (Chamow et al.,1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr OpinImmunol 9:195-200, both incorporated entirely by reference). “Fc fusion”is herein meant to be synonymous with the terms “immunoadhesin”, “Igfusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes)as used in the prior art (Chamow et al., 1996, Trends Biotechnol14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fcfusion is a protein wherein one or more polypeptides is operably linkedto Fc. An Fc fusion combines the Fc region of an antibody, and thus itsfavorable effector functions and pharmacokinetics, with thetarget-binding region of a receptor, ligand, or some other protein orprotein domain. The role of the latter is to mediate target recognition,and thus it is functionally analogous to the antibody variable region.Because of the structural and functional overlap of Fc fusions withantibodies, the discussion on antibodies in the present inventionextends also to Fc fusions.

Virtually any protein or small molecule may be linked to Fc to generatean Fc fusion. Protein fusion partners may include, but are not limitedto, the variable region of any antibody, the target-binding region of areceptor, an adhesion molecule, a ligand, an enzyme, a cytokine, achemokine, or some other protein or protein domain. Small moleculefusion partners may include any therapeutic agent that directs the Fcfusion to a therapeutic target. Such targets may be any molecule,preferably an extracellular receptor, that is implicated in disease.

In one embodiment, the Fc variants of the invention are antibody “fusionproteins”, sometimes referred to herein as “antibody conjugates”. Thefusion partner or conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antibody and on the conjugate partner. Conjugate andfusion partners may be any molecule, including small molecule chemicalcompounds and polypeptides. For example, a variety of antibodyconjugates and methods are described in Trail et al., 1999, Curr. Opin.Immunol. 11:584-588, incorporated entirely by reference. Possibleconjugate partners include but are not limited to cytokines, cytotoxicagents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenicagents, a tyrosine kinase inhibitors, and other therapeutically activeagents. In some embodiments, conjugate partners may be thought of moreas payloads, that is to say that the goal of a conjugate is targeteddelivery of the conjugate partner to a targeted cell, for example acancer cell or immune cell, by the Fc variant. Thus, for example, theconjugation of a toxin to an Fc variant targets the delivery of saidtoxin to cells expressing the target antigen. As will be appreciated byone skilled in the art, in reality the concepts and definitions offusion and conjugate are overlapping. The designation of a fusion orconjugate is not meant to constrain it to any particular embodiment ofthe present invention. Rather, these terms are used loosely to conveythe broad concept that any Fc variant of the present invention may belinked genetically, chemically, or otherwise, to one or morepolypeptides or molecules to provide some desirable property.

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs; for the latter, see U.S.2003/0050331, incorporated enirely by reference.

In one embodiment, the Fc variants of the present invention are fused orconjugated to a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. For example, as described inPenichet et al., 2001, J. Immunol. Methods 248:91-101, incorporatedentirely by reference, cytokines may be fused to antibody to provide anarray of desirable properties. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; C5a; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In an alternate embodiment, the Fc variants of the present invention arefused, conjugated, or operably linked to a toxin, including but notlimited to small molecule toxins and enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof. For example, a variety of immunotoxins and immunotoxinmethods are described in Thrush et al., 1996, Ann. Rev. Immunol.14:49-71, incorporated entirely by reference. Small molecule toxinsinclude but are not limited to calicheamicin, maytansine (U.S. Pat. No.5,208,020, incorporated entirely by reference), trichothene, and CC1065.In one embodiment of the invention, the Fc variant is conjugated to oneor more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al., 1992, Cancer Research 52: 127-131,incorporated entirely by reference) to generate a maytansinoid-antibodyconjugate. Another conjugate of interest comprises an Fc variantconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. Structural analogues ofcalicheamicin that may be used include but are not limited to γ₁ ¹, α₂¹, α₃, N-acetyl-γ₁ ¹, PSAG, and Θ¹ ₁, (Hinman et al., 1993, CancerResearch 53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928)(U.S. Pat. No. 5,714,586; U.S. Pat. No. 5,712,374; U.S. Pat. No.5,264,586; U.S. Pat. No. 5,773,001, all incorporated enirely byreference). Dolastatin 10 analogs such as auristatin E (AE) andmonomethylauristatin E (MMAE) may find use as conjugates for the Fcvariants of the present invention (Doronina et al., 2003, Nat Biotechnol21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65, bothincorporated entirely by reference). Useful enyzmatically active toxinsinclude but are not limited to diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.See, for example, PCT WO 93/21232, incorporated entirely by reference.The present invention further contemplates a conjugate between an Fcvariant of the present invention and a compound with nucleolyticactivity, for example a ribonuclease or DNA endonuclease such as adeoxyribonuclease (Dnase).

In an alternate embodiment, an Fc variant of the present invention maybe fused, conjugated, or operably linked to a radioisotope to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugate antibodies. Examples include, but are notlimited to, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, andradioactive isotopes of Lu. See for example, reference.

In yet another embodiment, an Fc variant of the present invention may beconjugated to a “receptor” (such streptavidin) for utilization in tumorpretargeting wherein the Fc variant-receptor conjugate is administeredto the patient, followed by removal of unbound conjugate from thecirculation using a clearing agent and then administration of a “ligand”(e.g. avidin) which is conjugated to a cytotoxic agent (e.g. aradionucleotide). In an alternate embodiment, the Fc variant isconjugated or operably linked to an enzyme in order to employ AntibodyDependent Enzyme Mediated Prodrug Therapy (ADEPT). ADEPT may be used byconjugating or operably linking the Fc variant to a prodrug-activatingenzyme that converts a prodrug (e.g. a peptidyl chemotherapeutic agent,see PCT WO 81/01145, incorporated entirely by reference) to an activeanti-cancer drug. See, for example, PCT WO 88/07378 and U.S. Pat. No.4,975,278, both incorporated entirely by reference. The enzyme componentof the immunoconjugate useful for ADEPT includes any enzyme capable ofacting on a prodrug in such a way so as to covert it into its moreactive, cytotoxic form. Enzymes that are useful in the method of thisinvention include but are not limited to alkaline phosphatase useful forconverting phosphate-containing prodrugs into free drugs; arylsulfataseuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase useful for converting non-toxic 5-fluorocytosine intothe anti-cancer drug, 5-fluorouracil; proteases, such as serratiaprotease, thermolysin, subtilisin, carboxypeptidases and cathepsins(such as cathepsins B and L), that are useful for convertingpeptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases,useful for converting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as .beta.-galactosidase andneuramimidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized with.alpha.-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, for example, Massey, 1987, Nature 328: 457-458, incorporatedentirely by reference). Fc variant-abzyme conjugates can be prepared fordelivery of the abzyme to a tumor cell population. A variety ofadditional conjugates are contemplated for the Fc variants of thepresent invention. A variety of chemotherapeutic agents, anti-angiogenicagents, tyrosine kinase inhibitors, and other therapeutic agents aredescribed below, which may find use as Fc variant conjugates.

Also contemplated as fusion and conjugate partners are Fc polypeptides.Thus an Fc variant may be a multimeric Fc polypeptide, comprising two ormore Fc regions. The advantage of such a molecule is that it providesmultiple binding sites for Fc receptors with a single protein molecule.In one embodiment, Fc regions may be linked using a chemical engineeringapproach. For example, Fab's and Fc's may be linked by thioether bondsoriginating at cysteine residues in the hinges, generating moleculessuch as FabFc₂. Fc regions may be linked using disulfide engineeringand/or chemical cross-linking. In a preferred embodiment, Fc regions maybe linked genetically. In a preferred embodiment, Fc regions in an Fcvariant are linked genetically to generated tandemly linked Fc regionsas described in U.S. Ser. No. 11/022,289, filed Dec. 21, 2004, entitled“Fc polypeptides with novel Fc ligand binding sites,” incorporatedentirely by reference. Tandemly linked Fc polypeptides may comprise twoor more Fc regions, preferably one to three, most preferably two Fcregions. It may be advantageous to explore a number of engineeringconstructs in order to obtain homo- or hetero- tandemly linked Fcvariants with the most favorable structural and functional properties.Tandemly linked Fc variants may be homo- tandemly linked Fc variants,that is an Fc variant of one isotype is fused genetically to another Fcvariant of the same isotype. It is anticipated that because there aremultiple Fc R, C1q, and/or FcRn binding sites on tandemly linked Fcpolypeptides, effector functions and/or pharmacokinetics may beenhanced. In an alternate embodiment, Fc variants from differentisotypes may be tandemly linked, referred to as hetero- tandemly linkedFc variants. For example, because of the capacity to target FcγR andFcaRI receptors, an Fc variant that binds both FcγRs and FcαRI mayprovide a significant clinical improvement.

Fusion and conjugate partners may be linked to any region of an Fcvariant of the present invention, including at the N- or C-termini, orat some residue in-between the termini. In a preferred embodiment, afusion or conjugate partner is linked at the N- or C-terminus of the Fcvariant, most preferably the N-terminus. A variety of linkers may finduse in the present invention to covalently link Fc variants to a fusionor conjugate partner. By “linker”, “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof, herein is meanta molecule or group of molecules (such as a monomer or polymer) thatconnects two molecules and often serves to place the two molecules in apreferred configuration. Linkers are known in the art; for example,homo- or hetero-bifunctional linkers as are well known (see, 1994 PierceChemical Company catalog, technical section on cross-linkers, pages155-200, incorporated entirely by reference). A number of strategies maybe used to covalently link molecules together. These include, but arenot limited to polypeptide linkages between N- and C-termini of proteinsor protein domains, linkage via disulfide bonds, and linkage viachemical cross-linking reagents. In one aspect of this embodiment, thelinker is a peptide bond, generated by recombinant techniques or peptidesynthesis. The linker peptide may predominantly include the followingamino acid residues: Gly, Ser, Ala, or Thr. The linker peptide shouldhave a length that is adequate to link two molecules in such a way thatthey assume the correct conformation relative to one another so thatthey retain the desired activity. Suitable lengths for this purposeinclude at least one and not more than 50 amino acid residues.Preferably, the linker is from about 1 to 30 amino acids in length, withlinkers of 1 to 20 amino acids in length being most preferred. Usefullinkers include glycine-serine polymers (including, for example, (GS)n,(GSGGS)n [SEQ ID NO. 16] (GGGGS)n [SEQ ID NO. 17] and (GGGS)n [SEQ IDNO. 18], where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers, as willbe appreciated by those in the art. Alternatively, a variety ofnonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers,that is may find use to link the Fc variants of the present invention toa fusion or conjugate partner, or to link the Fc variants of the presentinvention to a conjugate.

Nonhuman, Chimeric, Humanized, and Fully Human Antibodies

The variable region of an antibody, as is well known in the art, cancompose sequences from a variety of species. In some embodiments, theantibody variable region can be from a nonhuman source, including butnot limited to mice, rats, rabbits, camels, llamas, and monkeys. In someembodiments, the scaffold components can be a mixture from differentspecies. As such, if the antibody of the invention may be a chimericantibody and/or a humanized antibody. In general, both “chimericantibodies” and “humanized antibodies” refer to antibodies that combineregions from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse or other nonhumanspecies and the constant region(s) from a human.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. No. 5,693,762, incorporated entirely by reference.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein). Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc. Natl. Acad. Sci. USA 91:969-973. In one embodiment, the parentantibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, that is, to increase the affinity ofthe variable region for its target antigen. Other humanization methodsmay involve the grafting of only parts of the CDRs, including but notlimited to methods described in U.S. Ser. No. 09/810,502; Tan et al.,2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.169:3076-3084. Structure-based methods may be employed for humanizationand affinity maturation, for example as described in U.S. Ser. No.10/153,159 and related applications, all incorporated enirely byreference. In certain variations, the immunogenicity of the antibody isreduced using a method described in U.S. Ser. No. 11/004,590, entitled“Methods of Generating Variant Proteins with Increased Host StringContent and Compositions Thereof”, filed on Dec. 3, 2004, incorporatedentirely by reference.

In one embodiment, the antibody is a fully human antibody with at leastone modification as outlined herein. “Fully human antibody” or “completehuman antibody” refers to a human antibody having the gene sequence ofan antibody derived from a human chromosome with the modificationsoutlined herein. Fully human antibodies may be obtained, for example,using transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol8:455-458) or human antibody libraries coupled with selection methods(Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108).

Targets

The Fc variants of the present invention may be agonists, antagonists,neutralizing, inhibitory, or stimulatory. The Fc variant may find use inan antibody composition that is monoclonal or polyclonal. In a preferredembodiment, the Fc variants of the present invention are used to killtarget cells that bear the target antigen, for example cancer cells. Inan alternate embodiment, the Fc variants of the present invention areused to block, antagonize, or agonize the target antigen, for examplefor antagonizing a cytokine or cytokine receptor. In an alternatelypreferred embodiment, the Fc variants of the present invention are usedto block, antagonize, or agonize the target antigen and kill the targetcells that bear the target antigen.

Virtually any antigen may be targeted by the Fc variants of the presentinvention, including but not limited to proteins, subunits, domains,motifs, and/or epitopes belonging to the following list of targets:17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 AdenosineReceptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin B,Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, ActivinRIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS,ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1,ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1,APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCl, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpi, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Mud), MUC18, Muellerian-inhibitin substance, Mug, MuSK,NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),PIGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAILR2DRS, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2),TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors, etc.

Fc Receptor Binding Properties

The Fc variants of the present invention may be optimized for a varietyof Fc receptor binding properties. An Fc variant that is engineered orpredicted to display one or more optimized properties is herein referredto as an “optimized Fc variant”. Properties that may be optimizedinclude but are not limited to enhanced or reduced affinity for an FcγR.In a preferred embodiment, the Fc variants of the present invention areoptimized to possess enhanced affinity for a human activating FcγR,preferably FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb, mostpreferrably FcγRIIa and FcγRIIIa. In an alternately preferredembodiment, the Fc variants are optimized to possess reduced affinityfor the human inhibitory receptor FcγRIIb. These preferred embodimentsare anticipated to provide Fc polypeptides with enhanced therapeuticproperties in humans, for example enhanced effector function and greateranti-cancer potency. In other embodiments, Fc variants of the presentinvention provide enhanced affinity for one or more FcγRs, yet reducedaffinity for one or more other FcγRs. For example, an Fc variant of thepresent invention may have enhanced binding to FcγRI, FcγRIIa, and/orFcγRIIIa, yet reduced binding to FcγRIIb.

By “greater affinity” or “improved affinity” or “enhanced affinity” or“better affinity” than a parent Fc polypeptide, as used herein is meantthat an Fc variant binds to an Fc receptor with a significantly higherequilibrium constant of association (K_(A)) or lower equilibriumconstant of dissociation (K_(D)) than the parent Fc polypeptide when theamounts of variant and parent polypeptide in the binding assay areessentially the same. For example, the Fc variant with improved Fcreceptor binding affinity may display from about 5 fold to about 1000fold, e.g. from about 10 fold to about 500 fold improvement in Fcreceptor binding affinity compared to the parent Fc polypeptide, whereFc receptor binding affinity is determined, for example, as disclosed inthe Examples herein. Accordingly, by “reduced affinity” as compared to aparent Fc polypeptide as used herein is meant that an Fc variant bindsan Fc receptor with significantly lower K_(A) or higher K_(D) than theparent Fc polypeptide.

In a preferred embodiment of the invention, the Fc variants provideselectively enhanced affinity to one or more human activating receptorsrelative to the inhibitory receptor FcγRIIb. Selectively enhancedaffinity to an activating receptor relative to FcγRIIb means either thatthe Fc variant has improved affinity for the activating receptor ascompared to the parent Fc polypeptide but has reduced affinity forFcγRIIb as compared to the parent Fc polypeptide, or it means that theFc variant has improved affinity for both activating and inhibitoryreceptors as compared to the parent Fc polypeptide, however theimprovement in affinity is greater for the activating receptor than itis for FcγRIIb. The purpose of grouping both of these Fc receptorproperties together is that currently it is not known for cells thatexpress both activating and inhibitory receptors whetheractivation/inhibition is determined by the absolute threshold of FcγRIIbengagement, or by the relative engagement by activating and inhibitoryreceptors. The preferred application of Fc variants with such Fcreceptor affinity profiles is to impart antibodies, Fc fusions, or otherFc polypeptides with enhanced FcγR-mediated effector function andcellular activation, specifically for cells that express both activatingand inhibitory receptors including but not limited to neutrophils,monocytes and macrophages, and dendritic cells.

In alternately preferred embodiments of the present invention, the Fcvariants reduce or ablate binding to one or more FcγRs, reduce or ablatebinding to one or more complement proteins, reduce or ablate one or moreFcγR-mediated effector functions, and/or reduce or ablate one or morecomplement-mediated effector functions. In some embodiments, insertionsand/or deletions can be used to ablate the activity, and then amino acidsubstitutions can be used to increase binding, in many cases to one ormore selected FcγRs.

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the Fcvariants of the present invention. Whereas the specificity andselectivity of a given Fc variant for the different classes of FcγRssignficantly affects the capacity of an Fc variant to target a givenantigen for treatment of a given disease, the specificity or selectivityof an Fc variant for different polymorphic forms of these receptors mayin part determine which research or pre-clinical experiments may beappropriate for testing, and ultimately which patient populations may ormay not respond to treatment. Thus the specificity or selecitivty of Fcvariants of the present invention to Fc receptor polymorphisms,including but not limited to FcγRIIa, FcγRIIIa, and the like, may beused to guide the selection of valid research and pre-clinicalexperiments, clinical trial design, patient selection, dosingdependence, and/or other aspects concerning clinical trials.

Fc variants of the invention may comprise modifications that modulateinteraction with Fc receptors other than FcγRs, including but notlimited to complement proteins, FcRn, and Fc receptor homologs (FcRHs).FcRHs include but are not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5,and FcRH6 (Davis et al., 2002, Immunol. Reviews 190:123-136).

Clearly an important parameter that determines the most beneficialselectivity of a given Fc variant to treat a given disease is thecontext of the Fc variant. Thus the Fc receptor selectivity or specifityof a given Fc variant will provide different properties depending onwhether it composes an antibody, Fc fusion, or Fc variants with acoupled fusion or conjugate partner.

Preferably, the Fc receptor specificity of the Fc variant of the presentinvention will determine its therapeutic utility. The utility of a givenFc variant for therapeutic purposes will depend on the epitope or formof the target antigen and the disease or indication being treated. Forsome targets and indications, enhanced FcγR-mediated effector functionsmay be preferable. This may be particularly favorable for anti-cancer Fcvariants. Thus Fc variants may be used that comprise Fc variants thatprovide enhanced affinity for activating FcγRs and/or reduced affinityfor inhibitory FcγRs. For some targets and indications, it may befurther beneficial to utilize Fc variants that provide differentialselectivity for different activating FcγRs; for example, in some casesenhanced binding to FcγRIIa and FcγRIIIa may be desired, but not FcγRI,whereas in other cases, enhanced binding only to FcγRIIa may bepreferred. For certain targets and indications, it may be preferable toutilize Fc variants that enhance both FcγR-mediated andcomplement-mediated effector functions, whereas for other cases it maybe advantageous to utilize Fc variants that enhance either FcγR-mediatedor complement-mediated effector functions. For some targets or cancerindications, it may be advantageous to reduce or ablate one or moreeffector functions, for example by knocking out binding to C1q, one ormore FcγR's, FcRn, or one or more other Fc ligands. For other targetsand indications, it may be preferable to utilize Fc variants thatprovide enhanced binding to the inhibitory FcγRIIb, yet WT level,reduced, or ablated binding to activating FcγRs. This may beparticularly useful, for example, when the goal of an Fc variant is toinhibit inflammation or auto-immune disease, or modulate the immunesystem in some way.

In a preferred embodiment, the target of the Fc variants of the presentinvention is itself one or more Fc ligands. Fc polypeptides of theinvention can be utilized to modulate the activity of the immune system,and in some cases to mimic the effects of IVIg therapy in a morecontrolled, specific, and efficient manner. IVIg is effectively a highdose of immunoglobulins delivered intravenously. In general, IVIg hasbeen used to down-regulate autoimmune conditions. It has beenhypothesized that the therapeutic mechanism of action of IVIg involvesligation of Fc receptors at high frequency (J. Bayry et al., 2003,Transfusion Clinique et Biologique 10: 165-169; Binstadt et al., 2003,J. Allergy Clin. Immunol, 697-704). Indeed animal models of(thrombocytopenia purpura (ITP) show that the isolated Fc are the activeportion of IVIg (Samuelsson et al, 2001, Pediatric Research 50(5), 551).For use in therapy, iimmunoglobulins are harvested from thousands ofdonors, with all of the concomitant problems associated withnon-recombinant biotherapeutics collected from humans. An Fc variant ofthe present invention should serve all of the roles of IVIg while beingmanufactured as a recombinant protein rather than harvested from donors.

The immunomodulatory effects of IVIg may be dependent on productiveinteraction with one or more Fc ligands, including but not limited toFcγRs, complement proteins, and FcRn. In some embodiments, Fc variantsof the invention with enhanced affinity for FcγRIIb can be used topromote anti-inflammatory activity (Samuelsson et al., 2001, Science291: 484-486) and or to reduce autoimmunity (Hogarth, 2002, CurrentOpinion in Immunology, 14:798-802). In other embodiments, Fcpolypeptides of the invention with enhanced affinity for one or moreFcγRs can be utilized by themselves or in combination with additionalmodifications to reduce autoimmunity (Hogarth, 2002, Current Opinion inImmunology, 14:798-802). In alternative embodiments, Fc variants of theinvention with enhanced affinity for FcγRIIIa but reduced capacity forintracellular signaling can be used to reduce immune system activationby competitively interfering with FcγRIIIa binding. The context of theFc variant drammatically impacts the desired specificity. For example,Fc variants that provide enhanced binding to one or more activatingFcγRs may provide optimal immunomodulatory effects in the context of anantibody, Fc fusion, isolated Fc, or Fc fragment by acting as an FcγRantagonist (van Mirre et al., 2004, J. Immunol. 173:332-339). However,fusion or conjugation of two or more Fc variants may provide differenteffects, and for such an Fc polypeptide it may be optimal to utilize Fcvariants that provide enhanced affinity for an inhibitory receptor.

The Fc variants of the present invention may be used as immunomodulatorytherapeutics. Binding to or blocking Fc receptors on immune system cellsmay be used to influence immune response in immunological conditionsincluding but not limited to idiopathic thrombocytopenia purpura (ITP)and rheumatoid arthritis (RA) among others. By use of the affinityenhanced Fc variants of the present invention, the dosages required intypical IVIg applications may be reduced while obtaining a substantiallysimilar therapeutic effect. The Fc variants may provide enhanced bindingto an FcγR, including but not limited to FcγRIIa, FcγRIIb, FcγRIIIa,FcγRIIIb, and/or FcγRI. In particular, binding enhanements to FcγRIIbwould increase expression or inhibitory activity, as needed, of thatreceptor and improve efficacy. Alternatively, blocking binding toactivation receptors such as FcγRIIIb or FcγRI may improve efficacy. Inaddition, modulated affinity of the Fc variants for FcRn and/or alsocomplement may also provide benefits.

In one embodiment, Fc variants that provide enhanced binding to theinhibitory receptor FcγRIIb provide an enhancement to the IVIgtherapeutic approach. In particular, the Fc variants of the presentinvention that bind with greater affinity to the FcγRIIb receptor thanparent Fc polypeptide may be used. Such Fc variants would thus functionas FcγRIIb agonists, and would be expected to enhance the beneficialeffects of IVIg as an autoimmune disease therapeutic and also as amodulator of B-cell proliferation. In addition, such FcγRIIb-enhanced Fcvariants may also be further modified to have the same or limitedbinding to other receptors. In additional embodiments, the Fc variantswith enhanced FcγRIIb affinity may be combined with mutations thatreduce or ablate to other receptors, thereby potentially furtherminimizing side effects during therapeutic use.

Such immunomodulatory applications of the Fc variants of the presentinvention may also be utilized in the treatment of oncologicalindications, especially those for which antibody therapy involvesantibody-dependant cytotoxic mechanisms. For example, an Fc variant thatenhances affinity to FcγRIIb may be used to antagonize this inhibitoryreceptor, for example by binding to the Fc/FcγRIIb binding site butfailing to trigger, or reducing cell signaling, potentially enhancingthe effect of antibody-based anti-cancer therapy. Such Fc variants,functioning as FcγRIIb antagonists, may either block the inhibitoryproperties of FcγRIIb, or induce its inhibitory function as in the caseof IVIg. An FcγRIIb antagonist may be used as co-therapy in combinationwith any other therapeutic, including but not limited to antibodies,acting on the basis of ADCC related cytotoxicity. FcγRIIb antagonisticFc variants of this type are preferably isolated Fc or Fc fragments,although in alternate embodiments antibodies and Fc fusions may be used.

Glycoform Modifications

Many polypeptides, including antibodies, are subjected to a variety ofpost-translational modifications involving carbohydrate moieties, suchas glycosylation with oligosaccharides. There are several factors thatcan influence glycosylation. The species, tissue and cell type have allbeen shown to be important in the way that glycosylation occurs. Inaddition, the extracellular environment, through altered cultureconditions such as serum concentration, may have a direct effect onglycosylation (Lifely et al., 1995, Glycobiology 5(8): 813-822).

All antibodies contain carbohydrate at conserved positions in theconstant regions of the heavy chain. Each antibody isotype has adistinct variety of N-linked carbohydrate structures. Aside from thecarbohydrate attached to the heavy chain, up to 30% of human IgGs have aglycosylated Fab region. IgG has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate (Jefferiset al., 1998, Immunol. Rev. 163:59-76; Wright et al., 1997, TrendsBiotech 15:26-32). For human IgG, the core oligosaccharide normallyconsists of GlcNAc₂Man₃GlcNAc, with differing numbers of outer residues.

The carbohydrate moieties of the present invention will be describedwith reference to commonly used nomenclature for the description ofoligosaccharides. A review of carbohydrate chemistry which uses thisnomenclature is found in Hubbard et al. 1981, Ann. Rev. Biochem.50:555-583. This nomenclature includes, for instance, Man, whichrepresents mannose; GlcNAc, which represents 2-N-acetylglucosamine; Galwhich represents galactose; Fuc for fucose; and Glc, which representsglucose. Sialic acids are described by the shorthand notation NeuNAc,for 5-N-acetylneuraminic acid, and NeuNGc for 5-glycolylneuraminic.

The term “glycosylation” means the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to aglycoprotein. The oligosaccharide side chains are typically linked tothe backbone of the glycoprotein through either N- or O-linkages. Theoligosaccharides of the present invention occur generally are attachedto a CH2 domain of an Fc region as N-linked oligosaccharides. “N-linkedglycosylation” refers to the attachment of the carbohydrate moiety to anasparagine residue in a glycoprotein chain. The skilled artisan willrecognize that, for example, each of murine IgG1, IgG2a, IgG2b and IgG3as well as human IgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 domains have asingle site for N-linked glycosylation at amino acid residue 297 (Kabatet al. Sequences of Proteins of Immunological Interest, 1991).

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally consists of the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)₂ typical of biantennaryoligosaccharides. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region. A “bisecting GlcNAc” is a GlcNAc residueattached to the β1,4 mannose of the mature core carbohydrate structure.The bisecting GlcNAc can be enzymatically attached to the mature corecarbohydrate structure by a β(1,4)-N-acetylglucosaminyltransferase IIIenzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley etal., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to doso (Umana et al., 1999, Nature Biotech. 17:176-180).

The present invention contemplates Fc variants that comprise modifiedglycoforms or engineered glycoforms. By “modified glycoform” or“engineered glycoform” as used herein is meant a carbohydratecomposition that is covalently attached to a protein, for example anantibody, wherein said carbohydrate composition differs chemically fromthat of a parent protein. Engineered glycoforms may be useful for avariety of purposes, including but not limited to enhancing or reducingFcγR-mediated effector function. In a preferred embodiment, the Fcvariants of the present invention are modified to control the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region. A variety of methods are well known in theart for generating modified glycoforms (Umana et al., 1999, NatBiotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawaet al., 2003, J Biol Chem 278:3466-3473); (U.S. Pat. No. 6,602,684; U.S.Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1); (Potelligent™technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb™ glycosylationengineering technology [GLYCART biotechnology AG, Zurich, Switzerland];all of which are expressly incorporated by reference). These techniquescontrol the level of fucosylated and/or bisecting oligosaccharides thatare covalently attached to the Fc region, for example by expressing anIgG in various organisms or cell lines, engineered or otherwise (forexample Lec-13 CHO cells or rat hybridoma YB2/0 cells), by regulatingenzymes involved in the glycosylation pathway (for example FUT8[α1,6-fucosyltranserase] and/or β1-4-N-acetylglucosaminyltransferase III[GnTIII]), or by modifying carbohydrate(s) after the IgG has beenexpressed. The use of a particular mode of generating a modifiedglycoform, for example the use of the Lec-13 cell line in the presentstudy, is not meant to constrain the present invention to thatparticular embodiment. Rather, the present invention contemplates Fcvariants with modified glycoforms irrespective of how they are produced.

Other methods for modifying glycoforms of the Fc variants of theinvention include using glycoengineered strains of yeast (Li et al.,2006, Nature Biotechnology 24(2):210-215), moss (Nechansky et al., 2007,Mol Immunjol 44(7):1826-8), and plants (Cox et al., 2006, Nat Biotechnol24(12):1591-7). Methods for modying glycoforms include but are notlimited to using a glycoengineered strain of yeast Pichia pastoris (Liet al., 2006, Nature Biotechnology 24(2):210-215), a glycoengineeredstrain of the moss Physcomitrella patens wherein the enzymesβ1,2-xylosyltransferase and/or α1,3-fucosyltransferase are knocked outin (Nechansky et al., 2007, Mol Immunjol 44(7):1826-8), and the use ofRNA interference to inhibit endogenous alpha-1,3-fucosyltransferaseand/or beta-1,2-xylosyltransferase in the aquatic plant Lemna minor (Coxet al., 2006, Nat Biotechnol 24(12):1591-7).

Engineered glycoform typically refers to the different carbohydrate oroligosaccharide; thus for example an Fc variant may comprise anengineered glycoform. Alternatively, engineered glycoform may refer tothe Fc variant that comprises the different carbohydrate oroligosaccharide. For the purposes of modified glycoforms describedherein, a “parent antibody” is a glycosylated antibody having the sameamino acid sequence and mature core carbohydrate structure as anengineered glycoform of the present invention, except that fucose isattached to the mature core carbohydrate structure of the parentantibody. For instance, in a composition comprising the parentglycoprotein about 50-100% or about 70-100% of the parent glycoproteincomprises a mature core carbohydrate structure having fucose attachedthereto.

The present invention provides a composition comprising a glycosylatedFc variant having an Fc region, wherein about 51-100% of theglycosylated antibody in the composition comprises a mature corecarbohydrate structure which lacks fucose, attached to the Fc region ofthe antibody. More preferably, about 80-100% of the antibody in thecomposition comprises a mature core carbohydrate structure which lacksfucose and most preferably about 90-99% of the antibody in thecomposition lacks fucose attached to the mature core carbohydratestructure. In a most preferred embodiment, the antibody in thecomposition both comprises a mature core carbohydrate structure thatlacks fucose and additionally comprises at least one amino acidmodification in the Fc region. In the most preferred embodiment, thecombination of engineered glycoform and amino acid modification providesoptimal Fc receptor binding properties to the antibody.

Other Modifications

Fc variants of the present invention may comprise one or moremodifications that provide optimized properties that are notspecifically related to FcγR- or complement-mediated effector functionsper se. Said modifications may be amino acid modifications, or may bemodifications that are made enzymatically or chemically. Suchmodification(s) likely provide some improvement in the Fc variant, forexample an enhancement in its stability, solubility, function, orclinical use. The present invention contemplates a variety ofimprovements that made be made by coupling the Fc variants of thepresent invention with additional modifications.

In one embodiment, the variable region of an antibody of the presentinvention may be affinity matured, that is to say that amino acidmodifications have been made in the VH and/or VL domains of the antibodyto enhance binding of the antibody to its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Other improvements tothe target recognition properties may be provided by additionalmodifications. Such properties may include, but are not limited to,specific kinetic properties (i.e. association and dissociationkinetics), selectivity for the particular target versus alternativetargets, and selectivity for a specific form of target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of the targetantigen.

Fc variants of the invention may comprise one or more modifications thatprovide reduced or enhanced internalization of an Fc variant. In oneembodiment, Fc variants of the present invention can be utilized orcombined with additional modifications in order to reduce the cellularinternalization of an Fc variant that occurs via interaction with one ormore Fc ligands. This property might be expected to enhance effectorfunction, and potentially reduce immunogenicity of the Fc variants ofthe invention. Alternatively, Fc variants of the present invention canbe utilized directly or combined with additional modifications in orderto enhance the cellular internalization of an Fc variant that occurs viainteraction with one or more Fc ligands. For example, in a preferredembodiment, an Fc variant is used that provides enhanced binding toFcγRI, which is expressed on dendritic cells and active early in immuneresponse. This strategy could be further enhanced by combination withadditional modifications, either within the Fc variant or in an attachedfusion or conjugate partner, that promote recognition and presentationof Fc peptide fragments by MHC molecules. These strategies are expectedto enhance target antigen processing and thereby improve antigenicity ofthe target antigen (Bonnerot and Amigorena, 1999, Immunol Rev.172:279-84, incorporated entirely by reference), promoting an adaptiveimmune response and greater target cell killing by the human immunesystem. These strategies may be particularly advantageous when thetargeted antigen is shed from the cellular surface. An additionalapplication of these concepts arises with idiotype vaccineimmunotherapies, in which clone-specific antibodies produced by apatient's lymphoma cells are used to vaccinate the patient.

In a preferred embodiment, modifications are made to improve biophysicalproperties of the Fc variants of the present invention, including butnot limited to stability, solubility, and oligomeric state.Modifications can include, for example, substitutions that provide morefavorable intramolecular interactions in the Fc variant such as toprovide greater stability, or substitution of exposed nonpolar aminoacids with polar amino acids for higher solubility. A number ofoptimization goals and methods are described in U.S. Ser. No.10/379,392, incorporated entirely by reference, that may find use forengineering additional modifications to further optimize the Fc variantsof the present invention. The Fc variants of the present invention canalso be combined with additional modifications that reduce oligomericstate or size, such that tumor penetration is enhanced, or in vivoclearance rates are increased as desired.

Other modifications to the Fc variants of the present invention includethose that enable the specific formation or homodimeric orhomomultimeric molecules. Such modifications include but are not limitedto engineered disulfides, as well as chemical modifications oraggregation methods which may provide a mechanism for generatingcovalent homodimeric or homomultimers. For example, methods ofengineering and compositions of such molecules are described in Kan etal., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002,Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J. Immunol.148(9):2918-22, all incorporated enirely by reference. Additionalmodifications to the variants of the present invention include thosethat enable the specific formation or heterodimeric, heteromultimeric,bifunctional, and/or multifunctional molecules. Such modificationsinclude, but are not limited to, one or more amino acid substitutions inthe CH3 domain, in which the substitutions reduce homodimer formationand increase heterodimer formation. For example, methods of engineeringand compositions of such molecules are described in Atwell et al., 1997,J. Mol. Biol. 270(1):26-35, and Carter et al., 2001, J. Immunol. Methods248:7-15, both incorporated entirely by reference. Additionalmodifications include modifications in the hinge and CH3 domains, inwhich the modifications reduce the propensity to form dimers.

In further embodiments, the Fc variants of the present inventioncomprise modifications that remove proteolytic degradation sites. Thesemay include, for example, protease sites that reduce production yields,as well as protease sites that degrade the administered protein in vivo.In a preferred embodiment, additional modifications are made to removecovalent degradation sites such as deamidation (i.e. deamidation ofglutaminyl and asparaginyl residues to the corresponding glutamyl andaspartyl residues), oxidation, and proteolytic degradation sites.Deamidation sites that are particular useful to remove are those thathave enhance propensity for deamidation, including, but not limited toasparaginyl and gltuamyl residues followed by glycines (NG and QGmotifs, respectively). In such cases, substitution of either residue cansignificantly reduce the tendency for deamidation. Common oxidationsites include methionine and cysteine residues. Other covalentmodifications, that can either be introduced or removed, includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the “-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983), incorporated entirely by reference), acetylation ofthe N-terminal amine, and amidation of any C-terminal carboxyl group.Additional modifications also may include but are not limited toposttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The Fc variants of the present invention may comprise modifications thatinclude the use of unnatural amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci.U.S.A. 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin etal., 2003, Science 301(5635):964-7, all incorporated enirely byreference. In some embodiments, these modifications enable manipulationof various functional, biophysical, immunological, or manufacturingproperties discussed above. In additional embodiments, thesemodifications enable additional chemical modification for otherpurposes. Other modifications are contemplated herein. For example, theFc variant may be linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol. Additional amino acid modifications may be made to enablespecific or non-specific chemical or posttranslational modification ofthe Fc variants. Such modifications, include, but are not limited toPEGylation and glycosylation. Specific substitutions that can beutilized to enable PEGylation include, but are not limited to,introduction of novel cysteine residues or unnatural amino acids suchthat efficient and specific coupling chemistries can be used to attach aPEG or otherwise polymeric moiety. Introduction of specificglycosylation sites can be achieved by introducing novel N-X-T/Ssequences into the Fc variants of the present invention.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an anti-XXXXantibody of the present invention. See for example U.S. Ser. No.09/903,378, U.S. Ser. No. 10/754,296, U.S. Ser. No. 11/249,692, andreferences cited therein, all expressly incorporated by reference.

In particularly preferred embodiments of the invention, Fc variants ofthe present invention may be combined with Fc variants that alter FcRnbinding. Such variants may provide improved pharmacokinetic propertiesto the Fc variants of the invention. In particular, variants thatincrease Fc binding to FcRn include but are not limited to: 250E, 250Q,428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216, Hinton et al. 2006 Jounal of Immunology 176:346-356, U.S.Ser. No. 11/102,621, PCT/US2003/033037, PCT/US2004/011213, U.S. Ser. No.10/822,300, U.S. Ser. No. 10/687,118, PCT/US2004/034440, U.S. Ser. No.10/966,673 all entirely incorporated by reference), 256A, 272A, 286A,305A, 307A, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al,Journal of Biological Chemistry, 2001, 276(9):6591-6604, U.S. Ser. No.10/982,470, U.S. Pat. No. 6,737,056, U.S. Ser. No. 11/429,793, U.S. Ser.No. 11/429,786, PCT/US2005/029511, U.S. Ser. No. 11/208,422, allentirely incorporated by reference), 252F, 252T, 252Y, 252W, 254T, 256S,256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q,434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311 S (DallAcqua et al. Journal of Immunology, 2002, 169:5171-5180, U.S. Pat. No.7,083,784, PCT/US97/03321, U.S. Pat. No. 6,821,505, PCT/US01/48432, U.S.Ser. No. 11/397,328, all entirely incorporated by reference), 257C,257M, 257L, 257N, 257Y, 279E, 279Q, 279Y, insertion of Ser after 281,283F, 284E, 306Y, 307V, 308F, 308Y 311V, 385H, 385N, (PCT/US2005/041220,U.S. Ser. No. 11/274,065, U.S. Ser. No. 11/436,266 all entirelyincorporated by reference) 204D, 284E, 285E, 286D, and 290E(PCT/US2004/037929 entirely incorporated by reference).

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

In some embodiments, the covalent modification of the antibodies of theinvention comprises the addition of one or more labels. The term“labeling group” means any detectable label. In some embodiments, thelabeling group is coupled to the antibody via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in performing thepresent invention. In general, labels fall into a variety of classes,depending on the assay in which they are to be detected: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic labels(e.g., magnetic particles); c) redox active moieties; d) optical dyes;enzymatic groups (e.g. horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase); e) biotinylated groups; and f)predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags, etc.). In someembodiments, the labeling group is coupled to the antibody via spacerarms of various lengths to reduce potential steric hindrance. Variousmethods for labeling proteins are known in the art and may be used inperforming the present invention. Specific labels include optical dyes,including, but not limited to, chromophores, phosphors and fluorophores,with the latter being specific in many instances. Fluorophores can beeither “small molecule” fluores, or proteinaceous fluores. By“fluorescent label” is meant any molecule that may be detected via itsinherent fluorescent properties.

Production of Fc Variants

The present invention provides methods for producing and experimentallytesting Fc variants. The described methods are not meant to constrainthe present invention to any particular application or theory ofoperation. Rather, the provided methods are meant to illustrategenerally that one or more Fc variants may be produced andexperimentally tested to obtain Fc variants. General methods forantibody molecular biology, expression, purification, and screening aredescribed in Antibody Engineering, edited by Duebel & Kontermann,Springer-Verlag, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, CurrOpin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng2:339-76; Antibodies: A Laboratory Manual by Harlow & Lane, New York:Cold Spring Harbor Laboratory Press, 1988, all incorporated enirely byreference.

In one embodiment of the present invention, nucleic acids are createdthat encode the Fc variants, and that may then be cloned into hostcells, expressed and assayed, if desired. Thus, nucleic acids, andparticularly DNA, may be made that encode each protein sequence. Thesepractices are carried out using well-known procedures. For example, avariety of methods that may find use in the present invention aredescribed in Molecular Cloning—A Laboratory Manual, 3^(rd) Ed.(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), andCurrent Protocols in Molecular Biology (John Wiley & Sons), bothincorporated entirely by reference. As will be appreciated by thoseskilled in the art, the generation of exact sequences for a librarycomprising a large number of sequences is potentially expensive and timeconsuming. By “library” herein is meant a set of variants in any form,including but not limited to a list of nucleic acid or amino acidsequences, a list of nucleic acid or amino acid substitutions atvariable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising thevariant proteins, either in purified or unpurified form. Accordingly,there are a variety of techniques that may be used to efficientlygenerate libraries of the present invention. Such methods that may finduse in the present invention are described or referenced in U.S. Pat.No. 6,403,312; U.S. Ser. No. 09/782,004; U.S. Ser. No. 09/927,790; U.S.Ser. No. 10/218,102; PCT WO 01/40091; and PCT WO 02/25588, allincorporated entirely by reference. Such methods include but are notlimited to gene assembly methods, PCR-based method and methods which usevariations of PCR, ligase chain reaction-based methods, pooled oligomethods such as those used in synthetic shuffling, error-proneamplification methods and methods which use oligos with randommutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in the present invention forgenerating nucleic acids that encode Fc variants.

The Fc variants of the present invention may be produced by culturing ahost cell transformed with nucleic acid, preferably an expressionvector, containing nucleic acid encoding the Fc variants, under theappropriate conditions to induce or cause expression of the protein. Theconditions appropriate for expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. A wide varietyof appropriate host cells may be used, including but not limited tomammalian cells, bacteria, insect cells, and yeast. For example, avariety of cell lines that may find use in the present invention aredescribed in the ATCC® cell line catalog, available from the AmericanType Culture Collection.

In a preferred embodiment, the Fc variants are expressed in mammalianexpression systems, including systems in which the expression constructsare introduced into the mammalian cells using virus such as retrovirusor adenovirus. Any mammalian cells may be used, with human, mouse, rat,hamster, and primate cells being particularly preferred. Suitable cellsalso include known research cells, including but not limited to Jurkat Tcells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NSO cellsand variants thereof. In an alternately preferred embodiment, libraryproteins are expressed in bacterial cells. Bacterial expression systemsare well known in the art, and include Escherichia coli (E. coli),Bacillus subtilis, Streptococcus cremoris, and Streptococcus lividans.In alternate embodiments, Fc variants are produced in insect cells (e.g.Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S.cerevisiae, Pichia, etc). In an alternate embodiment, Fc variants areexpressed in vitro using cell free translation systems. In vitrotranslation systems derived from both prokaryotic (e.g. E. coli) andeukaryotic (e.g. wheat germ, rabbit reticulocytes) cells are availableand may be chosen based on the expression levels and functionalproperties of the protein of interest. For example, as appreciated bythose skilled in the art, in vitro translation is required for somedisplay technologies, for example ribosome display. In addition, the Fcvariants may be produced by chemical synthesis methods. Also transgenicexpression systems both animal (e.g. cow, sheep or goat milk,embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn,tobacco, duckweed, etc.)

The nucleic acids that encode the Fc variants of the present inventionmay be incorporated into an expression vector in order to express theprotein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in the present inventioninclude but are not limited to those which enable protein expression inmammalian cells, bacteria, insect cells, yeast, and in in vitro systems.As is known in the art, a variety of expression vectors are available,commercially or otherwise, that may find use in the present inventionfor expressing Fc variants.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the Fc variant, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

Fc variants may be operably linked to a fusion partner to enabletargeting of the expressed protein, purification, screening, display,and the like. Fusion partners may be linked to the Fc variant sequencevia a linker sequences. The linker sequence will generally comprise asmall number of amino acids, typically less than ten, although longerlinkers may also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS. A fusion partner may be a targeting or signal sequencethat directs Fc variant and any associated fusion partners to a desiredcellular location or to the extracellular media. As is known in the art,certain signaling sequences may target a protein to be either secretedinto the growth media, or into the periplasmic space, located betweenthe inner and outer membrane of the cell. A fusion partner may also be asequence that encodes a peptide or protein that enables purificationand/or screening. Such fusion partners include but are not limited topolyhistidine tags (His-tags) (for example H₆ and H₁₀ or other tags foruse with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g.Ni⁺² affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSPbiotinylation target sequence of the bacterial enzyme BirA, and epitopetags which are targeted by antibodies (for example c-myc tags,flag-tags, and the like). As will be appreciated by those skilled in theart, such tags may be useful for purification, for screening, or both.For example, an Fc variant may be purified using a His-tag byimmobilizing it to a Ni⁺² affinity column, and then after purificationthe same His-tag may be used to immobilize the antibody to a Ni⁺² coatedplate to perform an ELISA or other binding assay (as described below). Afusion partner may enable the use of a selection method to screen Fcvariants (see below). Fusion partners that enable a variety of selectionmethods are well-known in the art, and all of these find use in thepresent invention. For example, by fusing the members of an Fc variantlibrary to the gene III protein, phage display can be employed (Kay etal., Phage display of peptides and proteins: a laboratory manual,Academic Press, San Diego, Calif., 1996; Lowman et al., 1991,Biochemistry 30:10832-10838; Smith, 1985, Science 228:1315-1317,incorporated entirely by reference). Fusion partners may enable Fcvariants to be labeled. Alternatively, a fusion partner may bind to aspecific sequence on the expression vector, enabling the fusion partnerand associated Fc variant to be linked covalently or noncovalently withthe nucleic acid that encodes them. The methods of introducing exogenousnucleic acid into host cells are well known in the art, and will varywith the host cell used. Techniques include but are not limited todextran-mediated transfection, calcium phosphate precipitation, calciumchloride treatment, polybrene mediated transfection, protoplast fusion,electroporation, viral or phage infection, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei. In the case of mammalian cells, transfection may be eithertransient or stable.

In a preferred embodiment, Fc variants are purified or isolated afterexpression. Proteins may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of Fc variants. For example, the bacterial proteins A and Gbind to the Fc region. Likewise, the bacterial protein L binds to theFab region of some antibodies, as of course does the antibody's targetantigen. Purification can often be enabled by a particular fusionpartner. For example, Fc variants may be purified using glutathioneresin if a GST fusion is employed, Ni⁺² affinity chromatography if aHis-tag is employed, or immobilized anti-flag antibody if a flag-tag isused. For general guidance in suitable purification techniques, see,e.g. incorporated entirely by reference Protein Purification: Principlesand Practice, 3^(rd) Ed., Scopes, Springer-Verlag, NY, 1994,incorporated entirely by reference. The degree of purification necessarywill vary depending on the screen or use of the Fc variants. In someinstances no purification is necessary. For example in one embodiment,if the Fc variants are secreted, screening may take place directly fromthe media. As is well known in the art, some methods of selection do notinvolve purification of proteins. Thus, for example, if a library of Fcvariants is made into a phage display library, protein purification maynot be performed.

In Vitro Experimentation

Fc variants may be screened using a variety of methods, including butnot limited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label. The use offusion partners has been discussed above. By “labeled” herein is meantthat the Fc variants of the invention have one or more elements,isotopes, or chemical compounds attached to enable the detection in ascreen. In general, labels fall into three classes: a) immune labels,which may be an epitope incorporated as a fusion partner that isrecognized by an antibody, b) isotopic labels, which may be radioactiveor heavy isotopes, and c) small molecule labels, which may includefluorescent and colorimetric dyes, or molecules such as biotin thatenable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In a preferred embodiment, the functional and/or biophysical propertiesof Fc variants are screened in an in vitro assay. In vitro assays mayallow a broad dynamic range for screening properties of interest.Properties of Fc variants that may be screened include but are notlimited to stability, solubility, and affinity for Fc ligands, forexample FcγRs. Multiple properties may be screened simultaneously orindividually. Proteins may be purified or unpurified, depending on therequirements of the assay. In one embodiment, the screen is aqualitative or quantitative binding assay for binding of Fc variants toa protein or nonprotein molecule that is known or thought to bind the Fcvariant. In a preferred embodiment, the screen is a binding assay formeasuring binding to the target antigen. In an alternately preferredembodiment, the screen is an assay for binding of Fc variants to an Fcligand, including but are not limited to the family of FcγRs, theneonatal receptor FcRn, the complement protein C1q, and the bacterialproteins A and G. Said Fc ligands may be from any organism, with humans,mice, rats, rabbits, and monkeys preferred. Binding assays can becarried out using a variety of methods known in the art, including butnot limited to FRET (Fluorescence Resonance Energy Transfer) and BRET(Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreen™(Amplified Luminescent Proximity Homogeneous Assay), ScintillationProximity Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (SurfacePlasmon Resonance, also known as BIACORE®), isothermal titrationcalorimetry, differential scanning calorimetry, gel electrophoresis, andchromatography including gel filtration. These and other methods maytake advantage of some fusion partner or label of the Fc variant. Assaysmay employ a variety of detection methods including but not limited tochromogenic, fluorescent, luminescent, or isotopic labels.

The biophysical properties of Fc variants, for example stability andsolubility, may be screened using a variety of methods known in the art.Protein stability may be determined by measuring the thermodynamicequilibrium between folded and unfolded states. For example, Fc variantsof the present invention may be unfolded using chemical denaturant,heat, or pH, and this transition may be monitored using methodsincluding but not limited to circular dichroism spectroscopy,fluorescence spectroscopy, absorbance spectroscopy, NMR spectroscopy,calorimetry, and proteolysis. As will be appreciated by those skilled inthe art, the kinetic parameters of the folding and unfolding transitionsmay also be monitored using these and other techniques. The solubilityand overall structural integrity of an Fc variant may be quantitativelyor qualitatively determined using a wide range of methods that are knownin the art. Methods which may find use in the present invention forcharacterizing the biophysical properties of Fc variants include gelelectrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an Fc variant could be assayed for itsability to bind target antigen, then exposed to elevated temperature forone or more defined periods of time, then assayed for antigen bindingagain. Because unfolded and aggregated protein is not expected to becapable of binding antigen, the amount of activity remaining provides ameasure of the Fc variant's stability and solubility.

In a preferred embodiment, the library is screened using one or morecell-based or in vitro assays. For such assays, Fc variants, purified orunpurified, are typically added exogenously such that cells are exposedto individual variants or groups of variants belonging to a library.These assays are typically, but not always, based on the biology of theability of the Fc variant to bind to the target antigen and mediate somebiochemical event, for example effector functions like cellular lysis,phagocytosis, ligand/receptor binding inhibition, inhibition of growthand/or proliferation, apoptosisand the like. Such assays often involvemonitoring the response of cells to Fc variant, for example cellsurvival, cell death, cellular phagocytosis, cell lysis, change incellular morphology, or transcriptional activation such as cellularexpression of a natural gene or reporter gene. For example, such assaysmay measure the ability of Fc variants to elicit ADCC, ADCP, or CDC. Forsome assays additional cells or components, that is in addition to thetarget cells, may need to be added, for example serum complement, oreffector cells such as peripheral blood monocytes (PBMCs), NK cells,macrophages, and the like. Such additional cells may be from anyorganism, preferably humans, mice, rat, rabbit, and monkey. Crosslinkedor monomeric antibodies may cause apoptosis of certain cell linesexpressing the antibody's target antigen, or they may mediate attack ontarget cells by immune cells which have been added to the assay. Methodsfor monitoring cell death or viability are known in the art, and includethe use of dyes, fluorophores, immunochemical, cytochemical, andradioactive reagents. For example, caspase assays orannexin-flourconjugates may enable apoptosis to be measured, and uptakeor release of radioactive substrates (e.g. Chromium-51 release assays)or the metabolic reduction of fluorescent dyes such as alamar blue mayenable cell growth, proliferationor activation to be monitored. In apreferred embodiment, the DELFIA® EuTDA-based cytotoxicity assay (PerkinElmer, MA) is used. Alternatively, dead or damaged target cells may bemonitored by measuring the release of one or more natural intracellularproteins, for example lactate dehydrogenase. Transcriptional activationmay also serve as a method for assaying function in cell-based assays.In this case, response may be monitored by assaying for natural genes orproteins which may be upregulated or down-regulated, for example therelease of certain interleukins may be measured, or alternativelyreadout may be via a luciferase or GFP-reporter construct. Cell-basedassays may also involve the measure of morphological changes of cells asa response to the presence of an Fc variant. Cell types for such assaysmay be prokaryotic or eukaryotic, and a variety of cell lines that areknown in the art may be employed. Alternatively, cell-based screens areperformed using cells that have been transformed or transfected withnucleic acids encoding the Fc variants.

In vitro assays include but are not limited to binding assays, ADCC,CDC, cytotoxicity, proliferation, peroxide/ozone release, chemotaxis ofeffector cells, inhibition of such assays by reduced effector functionantibodies; ranges of activities such as >100× improvement or >100×reduction, blends of receptor activation and the assay outcomes that areexpected from such receptor profiles.

In Vivo Experimentation

The biological properties of the Fc variants of the present inventionmay be characterized in cell, tissue, and whole organism experiments. Asis know in the art, drugs are often tested in animals, including but notlimited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, in orderto measure a drug's efficacy for treatment against a disease or diseasemodel, or to measure a drug's pharmacokinetics, toxicity, and otherproperties. Said animals may be referred to as disease models. Withrespect to the Fc variants of the present invention, a particularchallenge arises when using animal models to evaluate the potential forin-human efficacy of candidate polypeptides—this is due, at least inpart, to the fact that Fc variants that have a specific effect on theaffinity for a human Fc receptor may not have a similar affinity effectwith the orthologous animal receptor. These problems can be furtherexacerbated by the inevitable ambiguities associated with correctassignment of true orthologues (Mechetina et al., Immunogenetics, 200254:463-468, incorporated entirely by reference), and the fact that someorthologues simply do not exist in the animal (e.g. humans possess anFcγRIIa whereas mice do not). Therapeutics are often tested in mice,including but not limited to nude mice, SCID mice, xenograft mice, andtransgenic mice (including knockins and knockouts). For example, an Fcvariant of the present invention that is intended as an anti-cancertherapeutic may be tested in a mouse cancer model, for example axenograft mouse. In this method, a tumor or tumor cell line is graftedonto or injected into a mouse, and subsequently the mouse is treatedwith the therapeutic to determine the ability of the Fc variant toreduce or inhibit cancer growth and metastasis. An alternative approachis the use of a SCID murine model in which immune-deficient mice areinjected with human PBLs, conferring a semi-functional and human immunesystem—with an appropriate array of human FcRs- to the mice that havesubsequently been injected with antibodies or Fc-polypeptides thattarget injected human tumor cells. In such a model, the Fc-polypeptidesthat target the desired antigen (such as her2/neu on SkOV3 ovariancancer cells) interact with human PBLs within the mice to engagetumoricidal effector functions. Such experimentation may providemeaningful data for determination of the potential of said Fc variant tobe used as a therapeutic. Any organism, preferably mammals, may be usedfor testing. For example because of their genetic similarity to humans,monkeys can be suitable therapeutic models, and thus may be used to testthe efficacy, toxicity, pharmacokinetics, or other property of the Fcvariants of the present invention. Tests of the Fc variants of thepresent invention in humans are ultimately required for approval asdrugs, and thus of course these experiments are contemplated. Thus theFc variants of the present invention may be tested in humans todetermine their therapeutic efficacy, toxicity, pharmacokinetics, and/orother clinical properties.

The Fc variants of the present invention may confer superior performanceon Fc-containing therapeutics in animal models or in humans. Thereceptor binding profiles of such Fc variants, as described in thisspecification, may, for example, be selected to increase the potency ofcytotoxic drugs or to target specific effector functions or effectorcells to improve the selectivity of the drug's action. Further, receptorbinding profiles can be selected that may reduce some or all effectorfunctions thereby reducing the side-effects or toxicity of suchFc-containing drug. For example, an Fc variant with reduced binding toFcγRIIIa, FcγRI and FcγRIIa can be selected to eliminate mostcell-mediated effector function, or an Fc variant with reduced bindingto C1q may be selected to limit complement-mediated effector functions.In some contexts, such effector functions are known to have potentialtoxic effects, therefore eliminating them may increase the safety of theFc-bearing drug and such improved safety may be characterized in animalmodels. In some contexts, such effector functions are known to mediatethe desirable therapeutic activity, therefore enhancing them mayincrease the activity or potency of the Fc-bearing drug and suchimproved activity or potency may be characterized in animal models.

Optimized Fc variants can be tested in a variety of orthotopic tumormodels. These clinically relevant animal models are important in thestudy of pathophysiology and therapy of aggressive cancers likepancreatic, prostate and breast cancer. Immune deprived mice including,but not limited to athymic nude or SCID mice are frequently used inscoring of local and systemic tumor spread from the site of intraorgan(e.g. pancreas, prostate or mammary gland) injection of human tumorcells or fragments of donor patients.

In preferred embodiments, Fc variants of the present invention may beassessed for efficacy in clinically relevant animal models of varioushuman diseases. In many cases, relevant models include varioustransgenic animals for specific tumor antigens.

Relevant transgenic models such as those that express human Fc receptors(e.g., CD16 including the gamma chain, FcγR1, RIIa/b, and others) couldbe used to evaluate and test Fc variant antibodies and Fc-fusions intheir efficacy. The evaluation of Fc variants by the introduction ofhuman genes that directly or indirectly mediate effector function inmice or other rodents that may enable physiological studies of efficacyin tumor toxicity or other diseases such as autoimmune disorders and RA.Human Fc receptors such as FcγRIIIa may possess polymorphisms such asthat in position 158 V or F which would further enable the introductionof specific and combinations of human polymorphisms into rodents. Thevarious studies involving polymorphism-specific FcRs is not limited tothis section, however encompasses all discussions and applications ofFcRs in general as specified in throughout this application. Fc variantsof the present invention may confer superior activity on Fc-containingdrugs in such transgenic models, in particular variants with bindingprofiles optimized for human FcγRIIIa mediated activity may showsuperior activity in transgenic CD16 mice. Similar improvements inefficacy in mice transgenic for the other human Fc receptors, e.g.FcγRIIa, FcγRI, etc., may be observed for Fc variants with bindingprofiles optimized for the respective receptors. Mice transgenic formultiple human receptors would show improved activity for Fc variantswith binding profiles optimized for the corresponding multiplereceptors.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides of the presentinvention may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules would preferably mimic—in the animalsystem—the FcR and/or complement biology of a corresponding candidatehuman Fc variant. This mimicry is most likely to be manifested byrelative association affinities between specific Fc variants and animalvs. human receptors. For example, if one were using a mouse model toassess the potential in-human efficacy of an Fc variant that hasenhanced affinity for human FcγRIIIa, an appropriate proxy variant wouldhave enhanced affinity for mouse FcγRIII-2 (mouse CD16-2). Alternativelyif one were using a mouse model to assess the potential in-humanefficacy of an Fc variant that has reduced affinity for the inhibitoryhuman FcγRIIb, an appropriate proxy variant would have reduced affinityfor mouse FcγRII. It should also be noted that the proxy Fc variantscould be created in the context of a human Fc variant, an animal Fcvariant, or both.

In a preferred embodiment, the testing of Fc variants may include studyof efficacy in primates (e.g. cynomolgus monkey model) to facilitate theevaluation of depletion of specific target cells harboring the targetantigen. Additional primate models include but not limited to that ofthe rhesus monkey and Fc polypetides in therapeutic studies ofautoimmune, transplantation and cancer.

Toxicity studies are performed to determine the antibody or Fc-fusionrelated-effects that cannot be evaluated in standard pharmacologyprofile or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity andcarcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherepeutics, naked or conjugated are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabeled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized and for which impurities/contaminants have been removed,that the test material is comparable throughout development, and GLPcompliance.

The pharmacokinetics (PK) of the Fc variants of the invention can bestudied in a variety of animal systems, with the most relevant beingnon-human primates such as the cynomolgus, rhesus monkeys. Single orrepeated i.v./s.c. administrations over a dose range of 6000-fold(0.05-300 mg/kg) can be evaluated for the half-life (days to weeks)using plasma concentration and clearance as well as volume ofdistribution at a steady state and level of systemic absorbance can bemeasured. Examples of such parameters of measurement generally includemaximum observed plasma concentration (Cmax), the time to reach Cmax(Tmax), the area under the plasma concentration-time curve from time 0to infinity [AUC(0-inf] and apparent elimination half-life (T1/2).Additional measured parameters could include compartmental analysis ofconcentration-time data obtained following i.v. administration andbioavailability. Examples of pharmacological/toxicological studies usingcynomolgus have been established for Rituxan and Zevalin in whichmonoclonal antibodies to CD₂₀ are cross-reactive. Biodistribution,dosimetry (for radiolabled antibodies), and PK studies can also be donein rodent models. Such studies would evaluate tolerance at all dosesadministered, toxicity to local tissues, preferential localization torodent xenograft animal models, depletion of target cells (e.g. CD20positive cells).

The Fc variants of the present invention may confer superiorpharmacokinetics on Fc-containing therapeutics in animal systems or inhumans. For example, increased binding to FcRn may increase thehalf-life and exposure of the Fc-containing drug. Alternatively,decreased binding to FcRn may decrease the half-life and exposure of theFc-containing drug in cases where reduced exposure is favorable such aswhen such drug has side-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof Fc variants of the present invention. Because Fc variants of thepresentation have varying affinities for the array of Fc receptors,further screening of the polypeptides for PD and/or PK properties may beextremely useful for defining the optimal balance of PD, PK, andtherapeutic efficacy conferred by each candidate polypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific tumor cells or blocking signaling mechanisms, measuringdepletion of target antigen expressing cells or signals, etc. The Fcvariants of the present invention may target particular effector cellpopulations and thereby direct Fc-containing drugs to recruit certainactivities to improve potency or to increase penetration into aparticularly favorable physiological compartment. For example,neutrophil activity and localization can be targeted by an Fc variantthat preferentially targets FcγRIIIb. Such pharmacodynamic effects maybe demonstrated in animal models or in humans.

Clinical Use

The Fc variants of the present invention may find use in a wide range ofproducts. In one embodiment the Fc variant of the present invention is atherapeutic, a diagnostic, or a research reagent, preferably atherapeutic. Alternatively, the Fc variants of the present invention maybe used for agricultural or industrial uses.

The Fc variants of the present invention may be used for varioustherapeutic purposes. As will be appreciated by those in the art, the Fcvariants of the present invention may be used for any therapeuticpurpose that antibodies, and the like may be used for. In a preferredembodiment, the Fc variants are administered to a patient to treatdisorders including but not limited to autoimmune and inflammatorydiseases, infectious diseases, and cancer.

A “patient” for the purposes of the present invention includes bothhumans and other animals, preferably mammals and most preferably humans.Thus the Fc variants of the present invention have both human therapyand veterinary applications. The term “treatment” or “treating” in thepresent invention is meant to include therapeutic treatment, as well asprophylactic, or suppressive measures for a disease or disorder. Thus,for example, successful administration of an Fc variant prior to onsetof the disease results in treatment of the disease. As another example,successful administration of an optimized Fc variant after clinicalmanifestation of the disease to combat the symptoms of the diseasecomprises treatment of the disease. “Treatment” and “treating” alsoencompasses administration of an optimized Fc variant after theappearance of the disease in order to eradicate the disease. Successfuladministration of an agent after onset and after clinical symptoms havedeveloped, with possible abatement of clinical symptoms and perhapsamelioration of the disease, comprises treatment of the disease. Those“in need of treatment” include mammals already having the disease ordisorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

In one embodiment, an Fc variant of the present invention isadministered to a patient having a disease involving inappropriateexpression of a protein or other molecule. Within the scope of thepresent invention this is meant to include diseases and disorderscharacterized by aberrant proteins, due for example to alterations inthe amount of a protein present, protein localization, posttranslationalmodification, conformational state, the presence of a mutant or pathogenprotein, etc. Similarly, the disease or disorder may be characterized byalterations molecules including but not limited to polysaccharides andgangliosides. An overabundance may be due to any cause, including butnot limited to overexpression at the molecular level, prolonged oraccumulated appearance at the site of action, or increased activity of aprotein relative to normal. Included within this definition are diseasesand disorders characterized by a reduction of a protein. This reductionmay be due to any cause, including but not limited to reduced expressionat the molecular level, shortened or reduced appearance at the site ofaction, mutant forms of a protein, or decreased activity of a proteinrelative to normal. Such an overabundance or reduction of a protein canbe measured relative to normal expression, appearance, or activity of aprotein, and said measurement may play an important role in thedevelopment and/or clinical testing of the Fc variants of the presentinvention.

By “cancer” and “cancerous” herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma),neuroendocrine tumors, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

More particular examples of such cancers include hematologicmalignancies, such as Hodgkin's lymphoma; non-Hodgkin's lymphomas(Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocyticleukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma,diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cellleukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursorcells, including B-cell acute lymphoblastic leukemia/lymphoma, andT-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of themature T and NK cells, including peripheral T-cell leukemias, adultT-cell leukemia/T-cell lymphomas and large granular lymphocyticleukemia, Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia; tumors of the central nervous system such as glioma,glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma,and retinoblastoma; solid tumors of the head and neck (eg.nasopharyngeal cancer, salivary gland carcinoma, and esophagael cancer),lung (eg. small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung),digestive system (eg. gastric or stomach cancer includinggastrointestinal cancer, cancer of the bile duct or biliary tract, coloncancer, rectal cancer, colorectal cancer, and anal carcinoma),reproductive system (eg. testicular, penile, or prostate cancer,uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer),skin (eg. melanoma, basal cell carcinoma, squamous cell cancer, actinickeratosis), liver (eg. liver cancer, hepatic carcinoma, hepatocellularcancer, and hepatoma), bone (eg. osteoclastoma, and osteolytic bonecancers) additional tissues and organs (eg. pancreatic cancer, bladdercancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer ofthe peritoneum, and Kaposi's sarcoma), and tumors of the vascular system(eg. angiosarcoma and hemagiopericytoma).

By “autoimmune diseases” herein include allogenic islet graft rejection,alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies(ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune myocarditis, autoimmuneneutropenia, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, autoimmune urticaria, Behcet's disease, bullouspemphigoid, cardiomyopathy, Castleman's syndrome, celiacspruce-dermatitis, chronic fatigue immune disfunction syndrome, chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn'sdisease, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis,glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture'ssyndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis,hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, IgM polyneuropathies, immune mediatedthrombocytopenia, juvenile arthritis, Kawasaki's disease, lichenplantus, lupus erthematosis, Meniere's disease, mixed connective tissuedisease, multiple sclerosis, type 1 diabetes mellitus, myastheniagravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobinulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld'sphenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis,scleroderma, Sjorgen's syndrome, solid organ transplant rejection,stiff-man syndrome, systemic lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, thrombotic thrombocytopeniapurpura, ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegner's granulomatosis.

By “inflammatory disorders” herein include acute respiratory distresssyndrome (ARDS), acute septic arthritis, adjuvant arthritis (Prakken etal., Springer Semin Immunopathol., 2003 August; 25(1):47-63,incorporated entirely by reference), juvenile idiopathic arthritis (deKleer et al., Arthritis Rheum. 2003 July; 47(7):2001-10, incorporatedentirely by reference), allergic encephalomyelitis, allergic rhinitis,allergic vasculitis, allergy, asthma, atherosclerosis, chronicinflammation due to chronic bacterial or viral infectionis, chronicobstructive pulmonary disease (COPD), coronary artery disease,encephalitis, inflammatory bowel disease, inflammatory osteolysis,inflammation associated with acute and delayed hypersensitivityreactions, inflammation associated with tumors, peripheral nerve injuryor demyelinating diseases, inflammation associated with tissue traumasuch as burns and ischemia, inflammation due to meningitis, multipleorgan injury syndrome, pulmonary fibrosis, sepsis and septic shock,Stevens-Johnson syndrome, undifferentiated arthropy, andundifferentiated spondyloarthropathy.

By “infectious diseases” herein include diseases caused by pathogenssuch as viruses, bacteria, fungi, protozoa, and parasites. Infectiousdiseases may be caused by viruses including adenovirus, cytomegalovirus,dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C,herpes simplex type I, herpes simplex type II, human immunodeficiencyvirus, (HIV), human papilloma virus (HPV), influenza, measles, mumps,papova virus, polio, respiratory syncytial virus, rinderpest,rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis,and the like. Infections diseases may also be caused by bacteriaincluding Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni,Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani,Diptheria, E. coli, Legionella, Helicobacter pylori, Mycobacteriumrickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S.pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersiniapestis, and the like. Infectious diseases may also be caused by fungisuch as Aspergillus fumigatus, Blastomyces dermatitidis, Candidaalbicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum, Penicillium marneffei, and the like. Infectious diseases mayalso be caused by protozoa and parasites such as chlamydia, kokzidioa,leishmania, malaria, rickettsia, trypanosoma, and the like.

Furthermore, antibodies of the present invention may be used to preventor treat additional conditions including but not limited to heartconditions such as congestive heart failure (CHF), myocarditis and otherconditions of the myocardium; skin conditions such as rosecea, acne, andeczema; bone and tooth conditions such as bone loss, osteoporosis,Paget's disease, Langerhans' cell histiocytosis, periodontal disease,disuse osteopenia, osteomalacia, monostotic fibrous dysplasia,polyostotic fibrous dysplasia, bone metastasis, bone pain management,humoral malignant hypercalcemia, periodontal reconstruction, spinal cordinjury, and bone fractures; metabolic conditions such as Gaucher'sdisease; endocrine conditions such as Cushing's syndrome; andneurological conditions.

A number of the receptors that may interact with the Fc variants of thepresent invention are polymorphic in the human population. For a givenpatient or population of patients, the efficacy of the Fc variants ofthe present invention may be affected by the presence or absence ofspecific polymorphisms in proteins. For example, FcγRIIIa is polymorphicat position 158, which is commonly either V (high affinity) or F (lowaffinity). Patients with the V/V homozygous genotype are observed tohave a better clinical response to treatment with the anti-CD20 antibodyRituxan® (rituximab), likely because these patients mount a stronger NKresponse (Dall'Ozzo et. al. (2004) Cancer Res. 64:4664-9, incorporatedentirely by reference). Additional polymorphisms include but are notlimited to FcγRIIa R131 or H131, and such polymorphisms are known toeither increase or decrease Fc binding and subsequent biologicalactivity, depending on the polymorphism. Fc variants of the presentinvention may bind preferentially to a particular polymorphic form of areceptor, for example FcγRIIIa 158 V, or to bind with equivalentaffinity to all of the polymorphisms at a particular position in thereceptor, for example both the 158V and 158F polymorphisms of FcγRIIIa.In a preferred embodiment, Fc variants of the present invention may haveequivalent binding to polymorphisms may be used in an antibody toeliminate the differential efficacy seen in patients with differentpolymorphisms. Such a property may give greater consistency intherapeutic response and reduce non-responding patient populations. Suchvariant Fc with identical binding to receptor polymorphisms may haveincreased biological activity, such as ADCC, CDC or circulatinghalf-life, or alternatively decreased activity, via modulation of thebinding to the relevant Fc receptors. In a preferred embodiment, Fcvariants of the present invention may bind with higher or lower affinityto one of the polymorphisms of a receptor, either accentuating theexisting difference in binding or reversing the difference. Such aproperty may allow creation of therapeutics particularly tailored forefficacy with a patient population possessing such polymorphism. Forexample, a patient population possessing a polymorphism with a higheraffinity for an inhibitory receptor such as FcγRIIb could receive a drugcontaining an Fc variant with reduced binding to such polymorphic formof the receptor, creating a more efficacious drug.

In a preferred embodiment, patients are screened for one or morepolymorphisms in order to predict the efficacy of the Fc variants of thepresent invention. This information may be used, for example, to selectpatients to include or exclude from clinical trials or, post-approval,to provide guidance to physicians and patients regarding appropriatedosages and treatment options. For example, in patients that arehomozygous or heterozygous for FcγRIIIa 158F antibody drugs such as theanti-CD₂₀ mAb, Rituximab are minimially effective (Carton 2002 Blood 99:754-758; Weng 2003 J. Clin. Oncol. 21:3940-3947, both incorporatedentirely by reference); such patients may show a much better clinicalresponse to the antibodies of the present invention. In one embodiment,patients are selected for inclusion in clinical trials for an antibodyof the present invention if their genotype indicates that they arelikely to respond significantly better to an antibody of the presentinvention as compared to one or more currently used antibodytherapeutics. In another embodiment, appropriate dosages and treatmentregimens are determined using such genotype information. In anotherembodiment, patients are selected for inclusion in a clinical trial orfor receipt of therapy post-approval based on their polymorphismgenotype, where such therapy contains an Fc variant engineered to bespecifically efficacious for such population, or alternatively wheresuch therapy contains an Fc variant that does not show differentialactivity to the different forms of the polymorphism.

Included in the present invention are diagnostic tests to identifypatients who are likely to show a favorable clinical response to an Fcvariant of the present invention, or who are likely to exhibit asignificantly better response when treated with an Fc variant of thepresent invention versus one or more currently used antibodytherapeutics. Any of a number of methods for determining FcγRpolymorphisms in humans known in the art may be used.

Furthermore, the present invention comprises prognostic tests performedon clinical samples such as blood and tissue samples. Such tests mayassay for effector function activity, including but not limited to ADCC,CDC, phagocytosis, and opsonization, or for killing, regardless ofmechanism, of cancerous or otherwise pathogenic cells. In a preferredembodiment, ADCC assays, such as those described previously, are used topredict, for a specific patient, the efficacy of a given Fc variant ofthe present invention. Such information may be used to identify patientsfor inclusion or exclusion in clinical trials, or to inform decisionsregarding appropriate dosages and treatment regemins. Such informationmay also be used to select a drug that contains a particular Fc variantthat shows superior activity in such assay.

Formulation

Pharmaceutical compositions are contemplated wherein an antibody of thepresent invention and one or more therapeutically active agents areformulated. Formulations of the antibodies of the present invention areprepared for storage by mixing said antibody having the desired degreeof purity with optional pharmaceutically acceptable carriers, excipientsor stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners andother flavoring agents; fillers such as microcrystalline cellulose,lactose, corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG). In a preferred embodiment, thepharmaceutical composition that comprises the antibody of the presentinvention may be in a water-soluble form, such as being present aspharmaceutically acceptable salts, which is meant to include both acidand base addition salts. “Pharmaceutically acceptable acid additionsalt” refers to those salts that retain the biological effectiveness ofthe free bases and that are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine. The formulations to beused for in vivo administration are preferably sterile. This is readilyaccomplished by filtration through sterile filtration membranes or othermethods.

The antibodies disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, ProcNatl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No.4,544,545; and PCT WO 97/38731, all incorporated entirely by reference.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556, incorporated entirely by reference. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent or other therapeuticallyactive agent is optionally contained within the liposome (Gabizon etal., 1989, J National Cancer Inst 81:1484, incorporated entirely byreference).

The antibody and other therapeutically active agents may also beentrapped in microcapsules prepared by methods including but not limitedto coacervation techniques, interfacial polymerization (for exampleusing hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacylate) microcapsules), colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed., 1980, incorporated entirely by reference. Sustained-releasepreparations may be prepared. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymer, which matrices are in the form of shaped articles, e.g. films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919, incorporatedentirely by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot® (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, andProLease® (commercially available from Alkermes), which is amicrosphere-based delivery system composed of the desired bioactivemolecule incorporated into a matrix of poly-DL-lactide-co-glycolide(PLG).

Administration

Administration of the pharmaceutical composition comprising an antibodyof the present invention, preferably in the form of a sterile aqueoussolution, may be done in a variety of ways, including, but not limitedto orally, subcutaneously, intravenously, intranasally, intraotically,transdermally, topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary, vaginally,parenterally, rectally, or intraocularly. In some instances, for examplefor the treatment of wounds, inflammation, etc., the antibody may bedirectly applied as a solution or spray. As is known in the art, thepharmaceutical composition may be formulated accordingly depending uponthe manner of introduction.

Subcutaneous administration may be preferable in some circumstancesbecause the patient may self-administer the pharmaceutical composition.Many protein therapeutics are not sufficiently potent to allow forformulation of a therapeutically effective dose in the maximumacceptable volume for subcutaneous administration. This problem may beaddressed in part by the use of protein formulations comprisingarginine-HCl, histidine, and polysorbate (see WO 04091658, incorporatedentirely by reference). Antibodies of the present invention may be moreamenable to subcutaneous administration due to, for example, increasedpotency, improved serum half-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The antibodies of the present invention may also bedelivered using such methods. For example, administration may venious beby intravenous infusion with 0.9% sodium chloride as an infusionvehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Antibodies of the present invention may bemore amenable to intrapulmonary delivery. FcRn is present in the lung,and may promote transport from the lung to the bloodstream (e.g.Syntonix WO 04004798, Bitonti et al. (2004) Proc. Nat. Acad. Sci.101:9763-8, both incorporated entirely by reference). Accordingly,antibodies that bind FcRn more effectively in the lung or that arereleased more efficiently in the bloodstream may have improvedbioavailability following intrapulmonary administration. Antibodies ofthe present invention may also be more amenable to intrapulmonaryadministration due to, for example, improved solubility or alteredisoelectric point.

Furthermore, antibodies of the present invention may be more amenable tooral delivery due to, for example, improved stability at gastric pH andincreased resistance to proteolysis. Furthermore, FcRn appears to beexpressed in the intestinal epithelia of adults (Dickinson et al. (1999)J. Clin. Invest. 104:903-11, incorporated entirely by reference), soantibodies of the present invention with improved FcRn interactionprofiles may show enhanced bioavailability following oraladministration. FcRn mediated transport of antibodies may also occur atother mucus membranes such as those in the gastrointestinal,respiratory, and genital tracts (Yoshida et al. (2004) Immunity20:769-83, incorporated entirely by reference).

In addition, any of a number of delivery systems are known in the artand may be used to administer the antibodies of the present invention.Examples include, but are not limited to, encapsulation in liposomes,microparticles, microspheres (eg. PLA/PGA microspheres), and the like.Alternatively, an implant of a porous, non-porous, or gelatinousmaterial, including membranes or fibers, may be used. Sustained releasesystems may comprise a polymeric material or matrix such as polyesters,hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamicacid and ethyl-L-gutamate, ethylene-vinyl acetate, lactic acid-glycolicacid copolymers such as the Lupron Depot®, andpoly-D-(−)-3-hydroxyburyric acid. It is also possible to administer anucleic acid encoding the antibody of the current invention, for exampleby retroviral infection, direct injection, or coating with lipids, cellsurface receptors, or other transfection agents. In all cases,controlled release systems may be used to release the antibody at orclose to the desired location of action.

Dosing

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active antibody in theformulation may vary from about 0.1 to 100 weight %. In a preferredembodiment, the concentration of the antibody is in the range of 0.003to 1.0 molar. In order to treat a patient, a therapeutically effectivedose of the antibody of the present invention may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. Dosages may range from 0.0001 to 100mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg ofbody weight, with 1 to 10 mg/kg being preferred.

In some embodiments, only a single dose of the antibody is used. Inother embodiments, multiple doses of the antibody are administered. Theelapsed time between administrations may be less than 1 hour, about 1hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours,about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.

In other embodiments the antibodies of the present invention areadministered in metronomic dosing regimes, either by continuous infusionor frequent administration without extended rest periods. Suchmetronomic administration may involve dosing at constant intervalswithout rest periods. Typically such regimens encompass chronic low-doseor continuous infusion for an extended period of time, for example 1-2days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lowerdoses may minimize side effects and the need for rest periods.

In certain embodiments the antibody of the present invention and one ormore other prophylactic or therapeutic agents are cyclicallyadministered to the patient. Cycling therapy involves administration ofa first agent at one time, a second agent at a second time, optionallyadditional agents at additional times, optionally a rest period, andthen repeating this sequence of administration one or more times. Thenumber of cycles is typically from 2-10. Cycling therapy may reduce thedevelopment of resistance to one or more agents, may minimize sideeffects, or may improve treatment efficacy.

Combination Therapies

The antibodies of the present invention may be administeredconcomitantly with one or more other therapeutic regimens or agents. Theadditional therapeutic regimes or agents may be used to improve theefficacy or safety of the antibody. Also, the additional therapeuticregimes or agents may be used to treat the same disease or a comorbidityrather than to alter the action of the antibody. For example, anantibody of the present invention may be administered to the patientalong with chemotherapy, radiation therapy, or both chemotherapy andradiation therapy. The antibody of the present invention may beadministered in combination with one or more other prophylactic ortherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,agents that promote proliferation of hematological cells, angiogenesisinhibitors, protein tyrosine kinase (PTK) inhibitors, additionalantibodies, FcγRIIb or other Fc receptor inhibitors, or othertherapeutic agents.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the antibody of thepresent invention and the other agent or agents are administered in asequence and within a time interval such that they may act together toprovide a benefit that is increased versus treatment with only eitherthe antibody of the present invention or the other agent or agents. Itis preferred that the antibody and the other agent or agents actadditively, and especially preferred that they act synergistically. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The skilled medical practitioner candetermine empirically, or by considering the pharmacokinetics and modesof action of the agents, the appropriate dose or doses of eachtherapeutic agent, as well as the appropriate timings and methods ofadministration.

In one embodiment, the antibodies of the present invention areadministered with one or more additional molecules comprising antibodiesor Fc. The antibodies of the present invention may be co-administeredwith one or more other antibodies that have efficacy in treating thesame disease or an additional comorbidity; for example two antibodiesmay be administered that recognize two antigens that are overexpressedin a given type of cancer, or two antigens that mediate pathogenesis ofan autoimmune or infectious disease.

Examples of anti-cancer antibodies that may be co-administered include,but are not limited to, anti-17-1A cell surface antigen antibodies suchas Panorex™ (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies;anti-A33 antibodies such as A33 and CDP-833; anti-α4β1 integrinantibodies such as natalizumab; anti-α4β7 integrin antibodies such asLDP-02; anti-α4β1 integrin antibodies such as F-200, M-200, and SJ-749;anti-αVβ3 integrin antibodies such as abciximab, CNTO-95, Mab-17E6, andVitaxin™ anti-complement factor 5 (C5) antibodies such as 5G1.1;anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodiessuch as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such asIDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B andOncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodiessuch as B43, MT-103, and Oncolysin B; anti-CD₂₀ antibodies such as 2H7,2H7.v16, 2H7.v114, 2H7.v115, Bexxar® (tositumomab, I-131 labeledanti-CD₂₀), Rituxan® (rituximab), and Zevalin® (Ibritumomab tiuxetan,Y-90 labeled anti-CD₂₀); anti-CD22 antibodies such as Lymphocide™(epratuzumab, Y-90 labeled anti-CD22); anti-CD23 antibodies such asIDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax®(daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30;anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin),Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodiessuch as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8,Antova™ and IDEC-131; anti-CD44 antibodies such as bivatuzumab;anti-CD46 antibodies; anti-CD52 antibodies such as Campath®(alemtuzumab); anti-CD55 antibodies such as SC-1; anti-CD56 antibodiessuch as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66eantibodies such as XR-303; anti-CD74 antibodies such as IMMU-110;anti-CD80 antibodies such as galiximab and IDEC-114; anti-CD89antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodiessuch as B-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148antibodies; anti-CEA antibodies such as cT84.66, labetuzumab, andPentacea™ anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4 antibodies;anti-EGFR antibodies such as ABX-EGF, Erbitux® (cetuximab), IMC-C225,and Merck Mab 425; anti-EpCAM antibodies such as Crucell's anti-EpCAM,ING-1, and IS-IL-2; anti-ephrin B2/EphB4 antibodies; anti-Her2antibodies such as Herceptin®, MDX-210; anti-FAP (fibroblast activationprotein) antibodies such as sibrotuzumab; anti-ferritin antibodies suchas NXT-211; anti-FGF-1 antibodies; anti-FGF-3 antibodies; anti-FGF-8antibodies; anti-FGFR antibodies, anti-fibrin antibodies; anti-G250antibodies such as WX-G250 and Rencarex®; anti-GD2 gangliosideantibodies such as EMD-273063 and TriGem; anti-GD3 gangliosideantibodies such as BEC2, KW-2871, and mitumomab; anti-gpIIb/IIIaantibodies such as ReoPro; anti-heparinase antibodies; anti-Her2/ErbB2antibodies such as Herceptin® (trastuzumab), MDX-210, and pertuzumab;anti-HLA antibodies such as Oncolym®, Smart 1D10; anti-HM1.24antibodies; anti-ICAM antibodies such as ICM3; anti-IgA receptorantibodies; anti-IGF-1 antibodies such as CP-751871 and EM-164;anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodies such asCNTO-328 and elsilimomab; anti-IL-15 antibodies such as HuMax™-IL15;anti-KDR antibodies; anti-laminin 5 antibodies; anti-Lewis Y antigenantibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies; anti-Mudantibodies such as BravaRex and TriAb; anti-NCAM antibodies such asERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragyn andTherex; anti-PSA antibodies; anti-PSCA antibodies such as IG8; anti-Ptkantbodies; anti-PTN antibodies; anti-RANKL antibodies such as AMG-162;anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such as MonopharmC; anti-STEAP antibodies; anti-TAG72 antibodies such as CC49-SCA andMDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-α antibodiessuch as CDP571, CDP870, D2E7, Humira® (adalimumab), and Remicade®(infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies; anti-VE-cadherin-2antibodies; and anti-VLA-4 antibodies such as Antegren™. Furthermore,anti-idiotype antibodies including but not limited to the GD3 epitopeantibody BEC2 and the gp72 epitope antibody 105AD7, may be used. Inaddition, bispecific antibodies including but not limited to theanti-CD3/CD20 antibody Bi20 may be used.

Examples of antibodies that may be co-administered to treat autoimmuneor inflammatory disease, transplant rejection, GVHD, and the likeinclude, but are not limited to, anti-α4β7 integrin antibodies such asLDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement(C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322,MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD11a antibodies,anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax,anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114,anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies suchas CDP850, anti-gpllb/IIIa antibodies such as ReoPro/Abcixima,anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740,anti-FcγR1 antibodies such as MDX-33, anti-IgE antibodies such asrhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodiessuch as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8,anti-interferon gamma antibodies, and anti-TNFa antibodies such asCDP571, CDP870, D2E7, Infliximab, MAK-195F, anti-VLA-4 antibodies suchas Antegren. Examples of other Fc-containing molecules that may beco-administered to treat autoimmune or inflammatory disease, transplantrejection, GVHD, and the like include, but are not limited to, the p75TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.

Examples of antibodies that may be co-administered to treat infectiousdiseases include, but are not limited to, anti-anthrax antibodies suchas ABthrax, anti-CMV antibodies such as CytoGam and sevirumab,anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G,anti-helicobacter antibodies such as Pyloran, anti-hepatitis Bantibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such asHRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab,RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb,BSYX-A110, and SE-Mab.

Alternatively, the antibodies of the present invention may beco-administered or with one or more other molecules that compete forbinding to one or more Fc receptors. For example, co-administeringinhibitors of the inhibitory receptor FcγRIIb may result in increasedeffector function. Similarly, co-administering inhibitors of theactivating receptors such as FcγRIIIa may minimize unwanted effectorfunction. Fc receptor inhibitors include, but are not limited to, Fcmolecules that are engineered to act as competitive inhibitors forbinding to FcγRIIb FcγRIIIa, or other Fc receptors, as well as otherimmunoglobulins and specificially the treatment called IVIg (intravenousimmunoglobulin). In one embodiment, the inhibitor is administered andallowed to act before the antibody is administered. An alternative wayof achieving the effect of sequential dosing would be to provide animmediate release dosage form of the Fc receptor inhibitor and then asustained release formulation of the antibody of the invention. Theimmediate release and controlled release formulations could beadministered separately or be combined into one unit dosage form.Administration of an FcγRIIb inhibitor may also be used to limitunwanted immune responses, for example anti-Factor VIII antibodyresponse following Factor VIII administration to hemophiliacs.

In one embodiment, the antibodies of the present invention areadministered with a chemotherapeutic agent. By “chemotherapeutic agent”as used herein is meant a chemical compound useful in the treatment ofcancer. Examples of chemotherapeutic agents include but are not limitedto alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan; androgenssuch as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); anti-metabolites such as methotrexate and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; folic acidreplenisher such as frolinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (suchas Tomudex); additional chemotherapeutics including aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;difluoromethylornithine (DMFO); elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide(VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; retinoic acid; esperamicins; capecitabine.Pharmaceutically acceptable salts, acids or derivatives of any of theabove may also be used.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug. By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery,” Directed Drug Delivery; andBorchardt et al., (ed.): 247-267, Humana Press, 1985, all incorporatedentirely by reference. The prodrugs that may find use with the presentinvention include but are not limited to phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, beta-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use with the antibodies of thepresent invention include but are not limited to any of theaforementioned chemotherapeutic agents.

A variety of other therapeutic agents may find use for administrationwith the antibodies of the present invention. In one embodiment, theantibody is administered with an anti-angiogenic agent. By“anti-angiogenic agent” as used herein is meant a compound that blocks,or interferes to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule or aprotein, for example an antibody, Fc fusion, or cytokine, that binds toa growth factor or growth factor receptor involved in promotingangiogenesis. The preferred anti-angiogenic factor herein is an antibodythat binds to Vascular Endothelial Growth Factor (VEGF). Other agentsthat inhibit signaling through VEGF may also be used, for exampleRNA-based therapeutics that reduce levels of VEGF or VEGF-R expression,VEGF-toxin fusions, Regeneron's VEGF-trap, and antibodies that bindVEGF-R. In an alternate embodiment, the antibody is administered with atherapeutic agent that induces or enhances adaptive immune response, forexample an antibody that targets CTLA-4. Additional anti-angiogenesisagents include, but are not limited to, angiostatin (plasminogenfragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin,bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor(CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatinA-4, endostatin (collagen XVIII fragment), farnesyl transferaseinhibitors, fibronectin fragment, gro-beta, halofuginone, heparinases,heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin(hCG), IM-862, interferon alpha, interferon beta, interferon gamma,interferon inducible protein 10 (IP-10), interleukin-12, kringle 5(plasminogen fragment), marimastat, metalloproteinase inhibitors (eg.TIMPs), 2-methodyestradiol, MMI 270 (CGS 27023A), plasminogen activiatorinhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16 kDafragment, proliferin-related protein (PRP), PTK 787/ZK 222594,retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248,tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1(TSP-1), TNP-470, transforming growth factor beta (TGF-β),vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

In a preferred embodiment, the antibody is administered with a tyrosinekinase inhibitor. By “tyrosine kinase inhibitor” as used herein is meanta molecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. Examples of such inhibitors include but are not limitedto quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline;pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g. those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering A G);pan-ErbB inhibitors such as C1-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (STI571, Gleevec®; Novartis); PKI 166(Novartis); GW2016 (Glaxo SmithKline); C1-1033 (Pfizer); EKB-569(Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11 (Imclone); or as described in any ofthe following patent publications: U.S. Pat. No. 5,804,396; PCT WO99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid); PCTWO 97/38983 (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCT WO99/06396 (Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO96/33978 (AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitinib (IRESSA™, ZD1839, AstraZeneca), and OSI-774(Tarceva™, OSI Pharmaceuticals/Genentech), all patent publicationsincorporated entirely by reference.

In another embodiment, the antibody is administered with one or moreimmunomodulatory agents. Such agents may increase or decrease productionof one or more cytokines, up- or down-regulate self-antigenpresentation, mask MHC antigens, or promote the proliferation,differentiation, migration, or activation state of one or more types ofimmune cells. Immunomodulatory agents include but not limited to:non-steroidal anti-inflammatory drugs (NSAIDs) such as asprin,ibuprofed, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin,ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib,naproxen, ketoprofen, and nabumetone; steroids (eg. glucocorticoids,dexamethasone, cortisone, hydroxycortisone, methylprednisolone,prednisone, prednisolone, trimcinolone, azulfidineicosanoids such asprostaglandins, thromboxanes, and leukotrienes; as well as topicalsteroids such as anthralin, calcipotriene, clobetasol, and tazarotene);cytokines such as TGFb, IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine,chemokine, or receptor antagonists including antibodies, solublereceptors, and receptor-Fc fusions against BAFF, B7, CCR2, CCR5, CD2,CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28,CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152,complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ,IFNγ, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3,MHC, selectins, TGFβ, TNFα, TNFβ, TNF-R1, T-cell receptor, includingEnbrel® (etanercept), Humira® (adalimumab), and Remicade® (infliximab);heterologous anti-lymphocyte globulin; other immunomodulatory moleculessuch as 2-amino-6-aryl-5 substituted pyrimidines, anti-idiotypicantibodies for MHC binding peptides and MHC fragments, azathioprine,brequinar, bromocryptine, cyclophosphamide, cyclosporine A,D-penicillamine, deoxyspergualin, FK506, glutaraldehyde, gold,hydroxychloroquine, leflunomide, malononitriloamides (eg. leflunomide),methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin,and sulfasasazine.

In an alternate embodiment, antibody of the present invention areadministered with a cytokine. By “cytokine” as used herein is meant ageneric term for proteins released by one cell population that act onanother cell as intercellular mediators. Examples of such cytokines arelymphokines, monokines, and traditional polypeptide hormones. Includedamong the cytokines are growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepaticgrowth factor; fibroblast growth factor; prolactin; placental lactogen;tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-beta; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-alpha, beta, and -gamma; colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosisfactor such as TNF-alpha or TNF-beta; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell culture,and biologically active equivalents of the native sequence cytokines.

In a preferred embodiment, cytokines or other agents that stimulatecells of the immune system are co-administered with the antibody of thepresent invention. Such a mode of treatment may enhance desired effectorfunction. For example, agents that stimulate NK cells, including but notlimited to IL-2 may be co-administered. In another embodiment, agentsthat stimulate macrophages, including but not limited to C5a, formylpeptides such as N-formyl-methionyl-leucyl-phenylalanine(Beigier-Bompadre et al. (2003) Scand. J. Immunol. 57: 221-8,incorporated entirely by reference), may be co-administered. Also,agents that stimulate neutrophils, including but not limited to G-CSF,GM-CSF, and the like may be administered. Furthermore, agents thatpromote migration of such immunostimulatory cytokines may be used. Alsoadditional agents including but not limited to interferon gamma, IL-3and IL-7 may promote one or more effector functions.

In an alternate embodiment, cytokines or other agents that inhibiteffector cell function are co-administered with the antibody of thepresent invention. Such a mode of treatment may limit unwanted effectorfunction.

In an additional embodiment, the antibody is administered with one ormore antibiotics, including but not limited to: aminoglycosideantibiotics (eg. apramycin, arbekacin, bambermycins, butirosin,dibekacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,ribostamycin, sisomycin, spectrinomycin), aminocyclitols (eg.sprctinomycin), amphenicol antibiotics (eg. azidamfenicol,chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics(eg. rifamide and rifampin), carbapenems (eg. imipenem, meropenem,panipenem); cephalosporins (eg. cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide,cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine),cephamycins (cefbuperazone, cefoxitin, cefminox, cefinetazole, andcefotetan); lincosamides (eg. clindamycin, lincomycin); macrolide (eg.azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin,tobramycin), monobactams (eg. aztreonam, carumonam, and tigernonam);mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam);penicillins (eg. amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, bexzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin O, penicillin V,penicillin V benzoate, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium); polypeptides (eg. bacitracin, colistin,polymixin B, teicoplanin, vancomycin); quinolones (amifloxacin,cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin,pipemidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin); rifampin; streptogramins (eg.quinupristin, dalfopristin); sulfonamides (sulfanilamide,sulfamethoxazole); tetracyclenes (chlortetracycline, demeclocyclinehydrochloride, demethylchlortetracycline, doxycycline, duramycin,minocycline, neomycin, oxytetracycline, streptomycin, tetracycline,vancomycin).

Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole,econazole, fluconazole, flucytosine, itraconazole, ketoconazole,niconazole, nystatin, terbinafine, terconazole, and tioconazole may alsobe used.

Antiviral agents including protease inhibitors, reverse transcriptaseinhibitors, and others, including type I interferons, viral fusioninhibitors, and neuramidase inhibitors, may also be used. Examples ofantiviral agents include, but are not limited to, acyclovir, adefovir,amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet,gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin,rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, andzidovudine, may be used.

The antibodies of the present invention may be combined with othertherapeutic regimens. For example, in one embodiment, the patient to betreated with an antibody of the present invention may also receiveradiation therapy. Radiation therapy can be administered according toprotocols commonly employed in the art and known to the skilled artisan.Such therapy includes but is not limited to cesium, iridium, iodine, orcobalt radiation. The radiation therapy may be whole body irradiation,or may be directed locally to a specific site or tissue in or on thebody, such as the lung, bladder, or prostate. Typically, radiationtherapy is administered in pulses over a period of time from about 1 to2 weeks. The radiation therapy may, however, be administered over longerperiods of time. For instance, radiation therapy may be administered topatients having head and neck cancer for about 6 to about 7 weeks.Optionally, the radiation therapy may be administered as a single doseor as multiple, sequential doses. The skilled medical practitioner candetermine empirically the appropriate dose or doses of radiation therapyuseful herein. In accordance with another embodiment of the invention,the antibody of the present invention and one or more other anti-cancertherapies are employed to treat cancer cells ex vivo. It is contemplatedthat such ex vivo treatment may be useful in bone marrow transplantationand particularly, autologous bone marrow transplantation. For instance,treatment of cells or tissue(s) containing cancer cells with antibodyand one or more other anti-cancer therapies, such as described above,can be employed to deplete or substantially deplete the cancer cellsprior to transplantation in a recipient patient.

It is of course contemplated that the antibodies of the invention mayemploy in combination with still other therapeutic techniques such assurgery or phototherapy.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation.

Example 1 Fc Variants with Reduced FcγR- and Complement-MediatedEffector Function

For some applications it may be favorable to reduce or eliminate bindingto one or more FcγRs, or reduce or eliminate one or more FcγR- orcomplement-mediated effector functions including but not limited toADCC, ADCP, and/or CDC. This is often the case for therapeuticantibodies whose mechanism of action involves blocking or antagonism butnot killing of the cells bearing target antigen. In these casesdepletion of target cells is undesirable and can be considered a sideeffect. Effector function can also be a problem for radiolabeledantibodies, referred to as radioconjugates, and antibodies conjugated totoxins, referred to as immunotoxins. These drugs can be used to destroycancer cells, but the recruitment of immune cells via Fc interactionwith FcγRs brings healthy immune cells in proximity to the deadlypayload (radiation or toxin), resulting in depletion of normal lymphoidtissue along with targeted cancer cells.

A previously unconsidered advantage of ablated FcγR- andcomplement-binding is that in cases where effector function is notneeded, binding to FcγR and complement may effectively reduce the activeconcentration of drug. Binding to Fc ligands may localize an antibody orFc fusion to cell surfaces or in complex with serum proteins wherein itis less active or inactive relative to when it is free (uncomplexed).This may be due to decreased effective concentration at binding siteswhere the antibody is desired, or perhaps Fc ligand binding may put theFc polypeptide in a conformation in which it is less active than itwould be if it were unbound. An additional consideration is thatFcγR-receptors may be one mechanism of antibody turnover, and canmediate uptake and processing by antigen presenting cells such asdendritic cells and macrophages. This may affect affect thepharmacokinetics (or in vivo half-life) of the antibody or Fc fusion andits immunogenicity, both of which are critical parameters of clinicalperformance.

Variants comprising insertions, deletions, and substitutions in the Fcregion were engineered to reduce or ablate interaction with FcγRs andcomplement. Insertions and deletions are not commonly used in proteinengineering strategies to modulate binding interactions because of thepotential for large perturbations to protein structure and stability.However as illustrated in FIG. 3, the flexible hinge region of anantibody may be uniquely amenable to engineering of insertions anddeletions. The hinge region, defined herein from position 221-236,contains part of the Fc region, and contains some binding determinantsfor interaction with Fc receptors. The FcγR binding site beginsapproximately at residue 233, yet structurally, the CH2 domain begins atposition 237. Thus it may be that insertions and deletions at andN-terminal to position 237 can be used to modulate interaction withFcγRs and complement, yet without affecting the stability and fidelityof the structured CH2 domain.

FIG. 4. lists a series of variants that were designed to reduce orablate interaction with FcγRs and complement. The variants wereconstructed in the context of an antibody comprising the Fv region ofthe anti-Her2 antibody trastuzumab and the constant heavy chain of thehuman IgG1. Human IgG2 and IgG4 versions were also constructed tocompare effector function of the Fc variants with naturally existing IgGantibodies.

The genes for the variable region of anti-Her2 antibody trastuzumab(Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-4289) wereconstructed using recursive PCR, and subcloned into the mammalianexpression vector pcDNA3.1Zeo (Invitrogen) comprising the full lengthlight kappa (Cκ) constant region for the light chain, or the heavy chainIgG1, IgG2, or IgG4 constant regions for the heavy chain. Amino acidmodifications were constructed in the Fc region of the antibodies in thepcDNA3.1Zeo vector using quick-change mutagenesis techniques(Stratagene). DNA was sequenced to confirm the fidelity of thesequences. Plasmids containing heavy chain gene (VH-CH1-CH2-CH3)(wild-type or variants) were co-transfected with plasmid containinglight chain gene (VL-Cκ) into 293T cells. Media were harvested 5 daysafter transfection, and antibodies were purified from the supernatantusing protein A affinity chromatography (Pierce). The sequences of theCK and IgG isotype constant chains are shown in FIG. 20.

In order to evaluate the interaction of the antibodies with Fcreceptors, the extracellular regions of human Fc receptors R131 and H131FcγRIIa, and V158 and F158 FcγRIIIa containing C-terminal 6×His tagswere obtained by PCR from clones obtained from the Mammalian GeneCollection (MGC), or generated de novo using recursive PCR. Receptorswere expressed in 293T cells and purified using nickel affinitychromatography. His-tagged extracellular regions of human FcγRI andFcγRIIb were obtained from R&D Systems.

Binding affinity to human FcγRs by Fc variant antibodies was measuredusing surface plasmon resonance (SPR), also referred to as BIAcore.Surface plasmon resonance measurements were performed using a Biacore3000 instrument (Biacore). Antibodies were captured onto an immobilizedprotein NG (Pierce) CM5 biosensor chip (Biacore), generated using astandard primary amine coupling protocol. All measurements wereperformed in HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%v/v surfactant P20, Biacore) and glycine buffer (10 mM glycine-HCl, pH1.5, Biacore) was used for the Protein A/G surface regeneration. Allantibodies (50 nM in HBS-EP) were immobilized on the protein A/G surfacefor 5 minutes at 1 ul/min. Fc receptors in serial dilutions wereinjected over antibody bound surface for 2 min at 20 ul/min followed by2 or 3 min dissociation phase. After each cycle the Protein A/G surfacewas regenerated by injecting glycine buffer (pH 1.5) for 30 s at 10ul/min. Data were processed by zeroing time and response before theinjection of receptor and by subtracting of appropriate non-specificsignals (response of reference channel and injection of running buffer).Kinetic analysis was performed by global fitting of binding data with a1:1 binding model (Langmuir) using the BlAevaluation software.

FIG. 5 a shows the normalized SPR sensorgrams for each concentration forbinding of WT IgG1 to the human Fc receptors FcγRI, both isoforms (H131and R131) FcγRIIa, FcγRIIb, and both isofoforms (V158 and F158) ofFcγRIIIa. An identical experiment was run for the other IgG isotypes(monoclonal IgG1, IgG2, and IgG4 with anti-Her2 variable region andpolyclonal serum IgG3 purchased commercially) as well as selectvariants. FIG. 5 b shows representative sensorgrams from each antibodyat the highest concentration for each receptor. The higher amplitude andslower off-rates observed with IgG1 and IgG3 relative to IgG2 and IgG4are consistent with the weaker binding of the latter. In contrast, nobinding was observed for all of the variants tested, with the exceptionof FcγRI for some of the variants. Langmuir fits of the Biacore data forall the variants provided equilibrium K_(D)s (FIG. 6). FIG. 7 shows aplot of the affinities (K_(A)=1/K_(D)) on a logarithmic scale forbinding of each antibody to each receptor. As can be seen, variantG236R/L328R shows no binding to any of the FcγRs. Variants L235G/G236R,N325A/L328R, and N325L/L328R show no binding to FcγRII and FcγRIIIreceptors, and show some albeit reduced binding to FcγRI.

Because binding to FcγRI was the most difficult among the Fc receptorsto reduce, this receptor was used as the primary screen for the othervariants. Other variants comprising insertions and deletions in thehinge region, as well as in some cases substitutions in the Fc region,were screened for binding to FcγRI using Biacore as described above.FIG. 8 shows the sensorgrams at the highest receptor concentration forall of the variants and WT IgG1. As can be seen, ̂236R, which has anarginine insertion after position 236, and G237#, which has a deletionof G237, have reduced but observable binding to FcγRI. In contrast, allother variants, comprising a variety of insertions and deletions in thehinge, as well as substitutions in the Fc region, have completelyablated binding to the high affinity receptor FcγRI. Select variantswere tested for binding to all signaling FcγRs by Biacore. FIG. 9 showssensorgrams at the highest concentration for binding of these variantsto human FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa. As can be seen, thesevariants show no detectable binding to any of the human FcγRs. Thebinding data for all of the variants to all of the receptors tested aresummarized in FIG. 6.

To assess the impact of the variants with reduced/ablated FcγR binding,select variants were tested for their capacity to mediate antibodydependent cellular cytotoxicity (ADCC). Human PBMCs were used aseffector cells, and the Her2+ cell line Sk-Br-3 was used as targetcells. PBMCs were purified from leukopacks using a Ficoll gradient, andADCC was measured by LDH release. Target cells were seeded into 96-wellplates and opsonized using native IgG or Fc variant antibodies at theindicated concentrations. Triton X100 and PBMCs alone were run ascontrols. Effector cells were added and plates were incubated at 37° C.,5% CO₂ for 4 h. Cells were then incubated with LDH reaction mixture for10 min, and fluorescence was measured using a Wallac Victor2 fluorometer(PerkinElmer). Fluorescence due to spontaneous PBMC and target celllysis (without antibodies) was subtracted from experimental values (withantibodies), normalized to maximal (Triton) and minimal (no Triton)lysis, and fit to a sigmoidal dose-response model. FIG. 10 shows thatvariants with reduced FcγR binding do not mediate ADCC, similarly to WTIgG2 and IgG4 and in contrast to WT IgG1 which binds with high affinityto FcγRs, particularly FcγRIIIa. PBMC ADCC is dominatetd by NK cells,which only express FcγRIIIa.

Monocyte-derived effector cells, including for example macrophages,express not only FcγRIIIa, but also FcγRI, FcγRIIa, and the inhibitoryreceptor FcγRIIb. Macrophages are phagocytes that act as scavengers toengulf dead cells, foreign substances, and other debris. Importantly,macrophages are profefssional antigen presenting cells (APCs), taking uppathogens and foreign structures in peripheral tissues, then migratingto secondary lymphoid organs to initiate adaptive immune responses byactivating naive T cells. Unlike NK cells, macrophages express the rangeof FcγRs, and thus their activation and function may be dependent onengagement of antibody immune complexes with receptors other than onlyFcγRIIIa. To evaluate the effect of ablation of FcγR affinity, thê236R/L328R variant was tested for its capacity to mediate macrophageantibody dependent cellular phagocytosis (ADCP). WT IgG1 was also run asa comparator and control.

Phagocytosis carried out using the variable region of an anti-CD19antibody, a humanized and affinity matured version of the murine 4G7antibody as described U.S. patent application Ser. No. 11/838,824,titled “Optimized Antibodies that Target CD19,” filed Aug. 14, 2007. Theheavy chain variable region of this antibody was subcloned into thepcDNA3.1 vector containing the heavy chain constant regions of IgG1 and̂236R/L328R. Antibodies were expressed and purified as described above.CD14⁺ macrophages were purified from PBMCs by EasySep® Human MonocyteEnrichment Kit without CD16 depletion (Stemcell Technologies). PurifiedCD14⁺ monocytes were cultured in M-CSF (Peprotech) at 50 ng/ml for 5days in a humidified incubator and differentiated into macrophages.Macrophage ADCP was determined by flow cytometry using CD19+ Ramos cellsas target cells. Target cells were labeled with PKH67 (Sigma) and seededinto 96-well plates in the presence of 10% human serum. Fc variantantibodies were diluted serially to half-log concentrations and added tothe target cells such that the highest concentration was 1 μg/ml.Monocyte-derived macrophages were then added at an effector to targetratio of 3:1, cells were spun down briefly, and incubated at 37° C. for4 h. Cells were detached from the plate surface with HyQtase, stainedwith anti-CD11b APC, anti-CD14 APC, and anti-CD66 PE, washed with PBS,and fixed with 1% paraformaldehyde. Phagocytosis was evaluated on a FACSCanto II flow cytometer (BD Biosciences), and percent phagocytosis wascalculated as the number of double positive cells divided by the totalnumber of tumor cells. The intensity of CD66 staining was used todetermine the degree to which tumor cells were internalized. FIG. 11shows the results of the experiment. As can be seen, in contrast to WTIgG1, the variant, which contains an insertion in the hinge and asubstitution in the Fc region, does not mediate ADCP.

Finally, select variants with reduced FcγR binding were further testedfor their capacity to mediate complement mediated cytotoxicity (CDC).The binding site for complement on the Fc region is separate from butoverlapping with the site for binding to FcγRs. CDC activity was testedin the context of antibodies targeting CD20. The variants wereconstructed in the context of the anti-CD₂₀ antibody PRO70769(PCT/US2003/040426, hereby entirely incorporated by reference), which isknown to mediate measurable CDC and ADCC in cell-based assays. The genesfor the variable regions of PRO70769 were constructed using recursivePCR, and subcloned into the mammalian expression vector pcDNA3.1Zeo(Invitrogen) comprising the full length light kappa (CK) for the lightchain, and either variant or WT IgG heavy chain constant regions.Antibodies were expressed and purified as described above. A cell-basedassay was used to measure the capacity of the Fc variants to mediateCDC. Lysis was measured using release of Alamar Blue to monitor lysis ofFc variant and WT anti-CD₂₀-opsonized WIL2-S lymphoma cells by humanserum complement. Target cells were washed 3× in 10% FBS medium bycentrifugation and resuspension, and WT or variant rituximab antibodywas added at the indicated final concentrations. Human serum complement(Quidel) was diluted 50% with medium and added to antibody-opsonizedtarget cells. Final complement concentration was ⅙^(th) original stock.Plates were incubated for 2 hrs at 37° C., Alamar Blue was added, cellswere cultured for two days, and fluorescence was measured. Data fromthis assay are shown in FIG. 12. As can be seen, the variants withmodifications at positions 235, 236, and 328, do not mediate CDCactivity, similarly to WT IgG2 and IgG4 and in contrast to IgG1anti-CD20.

The results show that insertions and deletions in the hinge region,particularly at or after positions 233-237, provide the capability toreduce and even ablate FcγR- and complement-mediated effector functions.In addition, the data show that combination of insertions and deletionswith substitutions in the Fc region are good. In particular, insertionsand deletions in the hinge region may be combined preferrably withsubstitutions at positions 235, 236, 237, 325, and 328. For example,substitutions 235G, 236R, 237K, 325L, 325A, and 328R may be combinedwith insertions after positions 233, 234, 235, 236, and 237, and/or withdeletions at positions 233, 234, 235, 236, and 237. Preferredembodiments of the invention for reducing or ablating FcγR- and/orcomplement-mediated effector function are provided in FIG. 13.

This list of preferred Fc variants is not meant to constrain the presentinvention. Because combinations of Fc variants of the present inventionhave typically resulted in additive or synergistic binding modulations,and accordingly additive or synergistic modulations in effectorfunction, it is anticipated that as yet unexplored combinations of theFc variants provided in the present invention, or with other previouslydisclosed modifications, will also provide favorable results. Indeed allcombinations of the any of the insertions, deletions, and/orsubstitutions provided are embodiments of the present invention.Furthermore, combinations of any of the Fc variants of the presentinvention with other discovered or undiscovered Fc variants may alsoprovide favorable properties, and these combinations are alsocontemplated as embodiments of the present invention. Further,insertions, deletions, and substitutions at all positions disclosedherein are contemplated.

As discussed above, reduced FcγR affinity and/or effector function maybe optimal for Fc polypeptides for which Fc ligand binding or effectorfunction leads to toxicity and/or reduced efficacy. For example,antibodies that target CTLA-4 block inhibition of T-cell activation andare effective at promoting anti-tumor immune response, but destructionof T cells via antibody mediated effector functions may becounterproductive to mechanism of action and/or potentially toxic.Indeed toxicity has been observed with clinical use of the anti-CTLA-4antibody ipilimumab (Maker et al., 2005, Ann Surg Oncol 12:1005-16,hereby entirely incorporated by reference). The sequences for theanti-CTLA-4 antibody ipilimumab (Mab 10D.1, MDX010) (U.S. Pat. No.6,984,720, hereby entirely incorporated by reference) are provided inFIG. 19. The use of an anti-CTLA-4 here is solely an example, and is notmeant to constrain application of the Fc variants to this antibody orany other particular Fc polypeptide. Other exemplary applications forreduced Fc ligand binding and/or effector function include but are notlimited to anti-TNFα antibodies, including for example infliximab andadalimumab, anti-VEGF antibodies, including for example bevacizumab,anti-α4-integrin antibodies, including for example natalizumab, andanti-CD32b antibodies, including for example those described in U.S.Ser. No. 10/643,857, hereby entirely incorporated by reference.

Example 2 Fc Variants with Selective FcγR Affinity

Improvement in affinity for FcγRs is a goal for enhancing thetherapeutic activity of antibodies that are used to treat cancers andinfectious diseases. A potentially important parameter in this approachis the selectivity of an antibody variant for activating versusinhibiting receptors. Whereas NK cells only express the activatingreceptor FcγRIIIa, other potentially important immune cell types,including neutrophils, macrophages, and dendritic cells, express theinhibitory receptor FcγRIIb, as well the other activating receptorsFcγRI and FcγRIIa. For these cell types optimal effector function mayresult from an antibody variant that has enhanced affinity foractivation receptors, for example FcγRI, FcγRIIa, and FcγRIIIa, yetreduced or unaltered affinity for the inhibitory receptor FcγRIIb.Notably, these other cells types can utilitize FcγRs to mediate not onlyinnate effector functions that directly lyse cells, for example ADCC,but can also phagocytose targeted cells and process antigen forpresentation to other immune cells, events that can ultimately lead tothe generation of adaptive immune response. Yet because all FcγRsinteract with the same binding site on Fc, and because of the highhomology among the FcγRs, obtaining variants that selectively enhance orreduce FcγR affinity is a major challenge.

The data provided in FIG. 7 indicate that WT IgG2 has a favorableFcγRIIa:FcγRIIb profile, that is greater affinity for the activatingreceptor H131 and R131FcγRIIa relative to the activating receptorFcγRIIb. However WT IgG2 has poor binding to FcγRI and FcγRIIIa. Aminoacid modifications were designed in an effort to engineer IgG2 such thatit maintains its favorable FcγRIIa:FcγRIIb profile, but binds the otheractivating receptors FcγRI and FcγRIIIa with enhanced affinity. Thesevariants, listed in FIG. 14, comprise insertions, deletions, andsubstitutions in the context of IgG2.

Variants were constructed in the context of the anti-Her2 antibodytrastuzumab, expressed, and purified as described above. Bindingaffinity to the human FcγRs was determined by Biacore as describedabove. Global langmuir fits of the data provided the equilibriumdissociation constants (K_(D)s) (FIG. 15 a). The fold affinities of theactivating receptors FcγRIIa and FcγRIIIa (both isoforms of each)relative to the inhibitory receptor FcγRIIb are plotted in FIG. 15 b.The log of the affinities and the ratio of activating to inhibitoryreceptors are plotted in FIG. 16 and FIG. 17 respectively. As can beseen, the insertions and deletions in the hinge region, as well assubstitutions in the Fc region, can be used to control the affinitiesand selectivities of the different FcγRs

Taken together, the data provided in the present invention indicate thatinsertions and deletions in the hinge region may be used to modulateFcγR affinity and selectivity. In particular, insertions after positions233, 234, 235, 236, and 237, and deletions at positions 233, 234, 235,236, and 237 may provide optimal effector function properties. Thecurrent invention also demonstrates that combination of said amino acidmodifications with other Fc substitutions may further provide optimaleffector function properties. For example, substitutions that may becombined with the modifications of the invention are described in U.S.Ser. No. 10/672,280; U.S. Ser. No. 10/822,231; U.S. Ser. No. 11/396,495;U.S. Ser. No. 11/124,620; U.S. Ser. No. 11/538,406; U.S. Pat. No.6,737,056; Shields et al, Journal of Biological Chemistry, 2001,276(9):6591-6604; U.S. Pat. No. 6,528,624; Idusogie et al., 2001, J.Immunology 166:2571-2572; U.S. Ser. No. 10/754,922; U.S. Ser. No.10/902,588; U.S. Ser. No. 10/370,749; Stavenhagen et al., 2007, CancerResearch 67(18):8882-90; all of which are herein expressly incorporatedby reference. In a most preferred embodiment, the insertions anddeletions of the invention are combined with one or more amino acidsubstutitions at a position selected from the group consisting of 234,235, 236, 239, 243, 247, 255, 267, 268, 270, 280, 292, 293, 295, 298,300, 305, 324, 326, 327, 328, 330, 332, 333, 334, 392, 396, and 421. Forexample, preferred substitutions that may be combined with theinsertions and deletions of the invention include but are not limited to234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D, 239E, 243L, 247L,255L, 267D, 267E, 267Q, 268D, 268E, 270E, 280H, 280Q, 280Y, 292P, 293R,295E, 298A, 298T, 298N, 300L, 305I, 324G, 324I, 326A, 326D, 326E, 326W,326Y, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, A330V, 332D, 332E, 333A,333S, 334A, 334L, 392T, 396L, and 421K. Preferred combinations ofinsertions, deletions, and substitutions are described in FIG. 18.

This list of preferred Fc variants is not meant to constrain the presentinvention. Indeed all combinations of the any of the insertions,deletions, and/or substitutions provided are embodiments of the presentinvention. Furthermore, combinations of any of the Fc variants of thepresent invention with other discovered or undiscovered Fc variants mayalso provide favorable properties, and these combinations are alsocontemplated as embodiments of the present invention. Further,insertions, deletions, and substitutions at all positions disclosedherein are contemplated.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

1. A composition comprising an antibody or immunoadhesin of a parent Fcpolypeptide, said antibody or immunoadhesin comprising an amino aciddeletion in a position selected from the group consisting of 233, 234,235 and 237 as compared to an IgG parent polypeptide, wherein numberingis according to the EU index.
 2. A composition comprising an antibody orimmunoadhesin of a parent Fc polypeptide, said antibody or immunoadhesincomprising an amino acid insertion in the Fc region of said parent Fcpolypeptide, wherein an amino acid is inserted after a position selectedfrom the group consisting of 233, 234, 235, 236, and 237, wherein saidinsertion is not a glycine after position 235, wherein numbering isaccording to the EU index.
 3. A composition comprising an antibody orimmunoadhesin of a parent Fc polypeptide, said antibody or immunoadhesincomprising an amino acid insertion in the Fc region of said parent Fcpolypeptide, wherein an amino acid is inserted after a position selectedfrom the group consisting of 233, 234, 235, 236, and 237, wherein saidantibody or immunoadhesin comprises an amino acid substitution at aposition selected from the group consisting of 221, 222, 224, 227, 228,230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244,245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 268, 269, 270,271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290,291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335336, 421 and 428, wherein numbering is according to the EU index.
 4. Acomposition according to claim 1 wherein said variant further comprisesan insertion at a position selected from the group consisting of 233,234, 235, 236 and
 237. 3. A composition according to claim 1 or 2,wherein said variant additionally comprises an amino acid substitution.4. A composition according to claim 3 wherein said amino acidsubstitution is at a position selected from the group consisting of 221,222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239,240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265,266, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283,285, 286, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302,313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335 336, 421 and
 428. 5. A composition according to claim4, wherein said substitution occurs at a position selected from thegroup consisting of 234, 235, 236, 239, 243, 247, 255, 267, 268, 270,280, 292, 293, 295, 298, 300, 305, 324, 326, 327, 328, 330, 332, 333,334, 392, 396, and
 421. 6. A composition according to claim 5 whereinsaid substitution occurs at a position selected from the groupconsisting of 235, 236, 237, 325, and
 328. 7. A composition according toclaim 6, wherein said substitution is selected from the group consistingof 235G, 236R, 237K, 325L, 325A, and 328R.
 8. A composition according toclaim 3 wherein said substitution is selected from the group consistingof 234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D, 239E, 243L,247L, 255L, 267D, 267E, 267Q, 268D, 268E, 270E, 280H, 280Q, 280Y, 292P,293R, 295E, 298A, 298T, 298N, 300L, 3051, 324G, 324I, 326A, 326D, 326E,326W, 326Y, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, A330V, 332D, 332E,333A, 333S, 334A, 334L, 392T, 396L, and 421 K.
 9. A compositionaccording to claim 1 or 2, wherein said antibody or immunoadhesin bindsto a target selected from the group consisting of CTLA-4, TNFα, VEGF,α4-integrin, or CD32b.
 10. A composition according to claim 1 or 2wherein said Fc variant is a human IgG1, IgG2, or IgG4 antibody.
 11. Acomposition according to claim 1 or 2 wherein said polypeptide is anantibody.
 12. A composition according to claim 11 wherein said antibodyis selected from the group consisting of a full-length antibody, anantibody fragment, and an Fc fusion protein.
 13. A composition accordingto claim 1 or 2 wherein said antibody is selected from the groupconsisting of human antibodies, humanized antibodies, and chimericantibodies.
 14. A composition according to claim 1 or 2 wherein saidpolypeptide is an engineered glycoform.
 15. A composition according toclaim 1 or 2, wherein said Fc variant exhibits altered binding to one ormore FcγRs.
 16. A composition according to claim 1 or 2 furthercomprising a pharmaceutical carrier.
 17. An isolated nucleic acidencoding an Fc variant according to claim 1 or
 2. 18. A methodcomprising administering a composition according to claim 1 or
 2. 19. Amethod according to claim 18 wherein said composition is administered toa subject having an indication selected from the group consisting of acancer, an autoimmune disorder, an infectious disease, or aninflammatory disorder.